Sp Family of Transcription Factors Is Involved in Valproic
Acid-induced Expression of G
i2*
Ifeanyi J.
Arinze
and
Yumiko
Kawai
From the Department of Biochemistry, Meharry Medical College,
Nashville, Tennessee 37208-3599
Received for publication, September 16, 2002, and in revised form, February 18, 2003
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ABSTRACT |
Valproic acid-induced gene expression has been
attributed to the DNA-binding activity of the transcription factor
activator protein 1 (AP-1). Using K562 cells, we have studied valproic
acid-induced transcription from the human G
i2 gene
promoter, which lacks AP-1-binding motifs. We find that valproic
acid-induced expression of G
i2 is inhibited by
mithramycin A, a compound that interferes with Sp1 binding to GC boxes
in DNA. Three Sp1-binding sequences, located at +68/+75,
50/
36, and
92/
85 in the promoter, accounted for about 60% of this
transcriptional effect, as judged by transient transfection assays.
Electrophoretic mobility shift assays indicated that these sites bind
members of the Sp family of transcription factors. Binding to DNA was
inhibited by mithramycin A and was greater in nuclear extracts from
cells treated with valproic acid than in control cells. Okadaic acid,
calyculin A, and fostriecin, which are potent inhibitors of protein
phosphatase, suppressed the transcriptional response to valproic
acid. This inhibitory effect was not observed when promoter constructs
containing mutations in the referenced Sp1-binding sites were used for
transfections. In nuclear extracts from cells cultured in the presence
of these inhibitors, the binding of Sp1/Sp3 to DNA probes was much less than in control cells. Alkaline phosphatase treatment of nuclear extracts resulted in enhanced binding of Sp proteins to the DNA probes.
These results are consistent with the idea that dephosphorylating conditions enhanced Sp binding to the DNA probes as well as Sp-mediated transcription induced by valproic acid. This study demonstrates that
the gene expression-inducing effect of valproic acid occurs, in part,
through the Sp family of transcription factors.
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INTRODUCTION |
Valproic acid (2-propylpentanoic acid), a branched
short-chain fatty acid, is an anticonvulsant agent that is also
clinically effective as a mood stabilizer in the treatment of manic
depression (bipolar affective disorder) (1-5). The biochemical basis
for the neurotrophic effects of valproic acid and other mood
stabilizers has intrigued investigators for a long time, but at the
present time the modes of action of these drugs are not clearly
understood (4, 6, 7). Among the effects of valproic acid is an increase in gene expression, an effect that has been demonstrated for the genes
for Bcl-2 and growth cone-associated p43 in SH-SY5Y cells (8),
Tcf/Lef-dependent transcription in 293T cells (9), and the
tyrosine hydroxylase gene (10, 11).
Reports that valproic acid increases the expression of genes regulated
by the transcription factor
AP-11 (12-14) have led to
the conclusion that the molecular mechanism of valproic acid-induced
gene expression is via DNA binding activity of this transcription
factor (8, 14). However, whether AP-1 is the only transcription factor
that can mediate valproic acid-induced gene expression is by no means
resolved. A gene promoter that lacks AP-1 motifs, such as the
G
i2 gene promoter (15-17), provides an excellent
paradigm for testing the role of other transcription factors. Using
K562 cells, we show, in this study, that valproic acid induces
expression of G
i2 and that it activates transcription from the G
i2 gene promoter, in part, through the Sp
family of transcription factors.
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EXPERIMENTAL PROCEDURES |
Chemicals and Reagents--
Sodium salts of caproic and valproic
acids were purchased from Sigma. Mithramycin A was purchased from
Biomol Research Laboratories, Inc. (Plymouth Meeting, PA). Okadaic
acid, calyculin A, and fostriecin were purchased from Alexis
Biochemicals (San Diego, CA). Immobilon-P polyvinylidene difluoride
transfer membranes for proteins were products of Millipore Corp.
(Bedford, MA). Oligonucleotides used for mutagenesis experiments were
purchased from Integrated DNA Technologies, Inc. (Coralville, IA).
Oligonucleotides used for gel shift assays and antibodies to
-tubulin, G
i2, Sp1, Sp2, Sp3, C/EBP
, and c-Fos
were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Anti-Sp1 antibody was also obtained from Upstate Biotechnology, Inc.
(Lake Placid, NY). Dual luciferase assay systems and calf intestinal
alkaline phosphatase were purchased from Promega (Madison, WI).
Expression plasmid harboring the gene for dominant negative ERK2 was
obtained from Dr. Melanie Cobb (University of Texas Southwestern
Medical School, Dallas, TX). The sources of all other
chemicals and reagents and the construction of the plasmid
containing full-length human G
i2 gene promoter (pG
i2(
1214/+115)-luc) and mutants thereof have been
previously described (18).
Cell Cultures and Transfection Studies--
K562 cells, obtained
from the American Type Culture Collection (Manassas, VA), were
maintained in culture as previously described (19). The cells (1 × 105 cells/well in 1 ml of medium) were seeded in 24-well
plates; wells on the periphery of the plates were not used. After
24 h, the cells were transfected with 0.5 µg of plasmid DNA
(containing the G
i2 gene promoter or mutant) and 1.5 µl of FuGENETM 6 transfection reagent (Roche Molecular
Biochemicals) 1 h before the addition of valproic or caproic
acid. In co-transfection experiments described in the legend to
Fig. 3, only 0.25 µg of the reporter plasmid was used. When chemical
inhibitors such as mithramycin A and okadaic acid were used, they were
added 1 h prior to the addition of valproic acid. The cells were
harvested 24 h later, by centrifugation at 12,000 × g (45 s) in 1.5-ml microcentrifuge tubes and washed once
with 1 ml of 1× phosphate-buffered saline, pH 7.4. The cell pellets
were then lysed with 150 µl of 1× cell lysis reagent (Promega,
Madison, WI) and kept at room temperature for 10 min. The lysed
material was then centrifuged for 2 min at 12,000 × g, and the luciferase activity and protein content of each
lysate were measured as previously described (18). In carrying out the
dual luciferase assay protocol, we noted that valproic acid robustly
induced the luciferase gene in conventional luc-reporter constructs
(e.g. Promega's pRL-TK-luc and pRL-CMV-luc reporter
vectors) usually used for normalization in transfection experiments,
making it impractical to use such plasmids for this purpose. This
problem was not encountered with promoterless constructs (e.g. phRG-B Renilla luciferase reporter vector
from Promega). With such constructs, the dual luciferase assay resulted
in similar -fold activation of transcription as when the luciferase
activities were normalized to the protein content of the samples.
Therefore, luciferase activity was routinely normalized to the protein
content of each sample, after correcting for basal activity of
GL3-basic and is expressed as -fold effect over cells that were not
treated with valproic acid.
Western Immunoblotting--
Immunoblotting of G
i2
was carried out with previously described protocols (18, 19). The
samples used for these assays were the same cell lysates prepared for
the luciferase assay. The same blots were reprobed for
-tubulin, as
loading control, to verify efficiency of transfer of protein to the
polyvinylidene difluoride membrane. Immunoblotting of Sp1, Sp2, and Sp3
in nuclear extracts was carried out with 10 µg of nuclear extract
protein; the blots were reprobed for C/EBP
and c-Fos, as loading controls.
Northern Blot Analysis--
Total RNA was isolated
with TRI REAGENTTM (Sigma) according to the manufacturer.
Northern blot analysis was performed as described previously, using
32P-labeled G
i2-specific oligonucleotide
probe (19). To monitor differences in loading and transfer among
samples, blots were stripped and rehybridized with an end-labeled
oligonucleotide complementary to the human 28 S rRNA.
Preparation of Nuclear Extracts and Electrophoretic Mobility
Shift Assays (EMSAs)--
Nuclear extracts were prepared as described
previously (18). For EMSA, annealed 5'-overhang oligonucleotide
sequence, containing Sp1-binding motif, was labeled with
[
-32P]dCTP, using the Klenow fill-in reaction, and
purified (18). The labeled probe was used for the EMSA reaction as
described in the legends to Figs. 5, 7, and 9. After electrophoresis,
the gel was dried and then exposed to Eastman Kodak Co. XAR5 film at
80 °C. The radiolabeled bands were detected by autoradiography.
Site-directed Mutagenesis--
Specific nucleotides in the
G
i2 gene promoter were mutated or deleted by using
QuikChangeTM mutagenesis kit from Stratagene, Inc. (La
Jolla, CA). Mutations were confirmed by DNA sequencing.
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RESULTS |
Valproic Acid Induces Transcription from the Human
G
i2 Gene Promoter--
We measured the transcriptional
effect of valproic acid by reporter gene assay, using the full-length
human G
i2 gene promoter linked to a luciferase reporter
gene, pG
i2(
1214/+115)-luc, transfected into K562
cells. For comparison, this parameter was also measured in cells
treated with another short-chain fatty acid (i.e. caproic acid (hexanoic acid)). Valproic acid caused a
dose-dependent increase in transcription from the
G
i2 gene promoter, whereas caproic acid had very little
effect compared with valproic acid (Fig. 1A). We assessed expression of
the endogenous G
i2 gene by using Western blotting assays
to measure G
i2 protein levels and Northern blotting
assays to measure G
i2 mRNA levels. As shown in Fig. 1B, increasing concentrations of valproic acid resulted in
corresponding increases in G
i2 levels that were about
4-, 6-, and 10-fold at 1, 2, and 5 mM valproate,
respectively. Valproic acid also induced a severalfold increase in
G
i2 mRNA levels (Fig. 1C, left
panel), and a dose-dependent increase was evident
(Fig. 1C, right panel). Taken together, the data
in Fig. 1 demonstrate that valproic acid induces expression of
G
i2 not only from the transfected promoter but also from
the endogenous promoter.

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Fig. 1.
Effects of valproic and caproic acids on the
expression of G i2. K562 cells
(1 × 105 cells/well in 1 ml of medium) were grown in
24-well plates for 24 h and then incubated with varying
concentrations of valproic or caproic acid for another 24 h, as
described under "Experimental Procedures." A,
transcription from the G i2 gene promoter:
dose-dependent effects of valproic and caproic acids. K562
cells were transfected with pG i2( 1214/+115)-luc,
treated with valproic or caproic acid, and harvested 24 h later.
Luciferase activities were normalized to protein content of each sample
and are expressed as -fold stimulation, relative to cells that were not
treated with either valproic or caproic acid. All promoter activities
were corrected for basal activity of GL3-basic. Values shown are
means ± S.E. for triplicate assays from four different cell
cultures. B, dose-dependent effects of valproic
and caproic acids on G i2 levels. G i2
levels were determined by Western blotting, using 10 µg of protein
for each sample. The same blot was reprobed for -tubulin as a
loading control. The data are representative of two similar Western
blots. C, G i2 mRNA levels (measured by
Northern blots) in control cells and cells treated with valproic acid.
G i2 mRNA levels were measured at 15, 20, and 24 h after treatment of the cells with 5 mM valproic acid
(left panel). The right panel shows
dose-dependent effects of valproic acid; for this analysis,
the cells were harvested for RNA preparation 24 h after the
addition of valproic acid to the cultures. For both panels,
the blots were stripped and rehybridized with an end-labeled
oligonucleotide complementary to the human 28 S rRNA, in order to
monitor differences in loading and transfer among samples. The data are
representative of two experiments. VPA, valproic acid;
CPA, caproic acid.
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Mithramycin A Inhibits Valproic Acid-induced G
i2
Gene Expression--
Because we have previously shown that
trichostatin A, a histone deacetylase inhibitor, induces transcription
from the G
i2 gene promoter (18), we were intrigued by
the report by Phiel et al. (9) that valproic acid mimics
trichostatin A in its ability to inhibit histone deacetylase. The
transcriptional action of trichostatin A is known to involve Sp1
(20-22). Therefore, we tested whether mithramycin A, a potent
inhibitor of Sp1 binding to GC boxes in DNA, which is capable of
interfering with Sp1-mediated gene transcription (23-27), would alter
valproic acid-induced G
i2 gene expression. First, we
tested whether mithramycin A would influence expression of the
endogenous gene by assessing G
i2 levels via
immunoblotting assays. Second, we tested whether mithramycin A would
influence promoter activity of transfected G
i2 promoter. For comparison, butyrate was included as a positive control in these
experiments, because we previously showed that butyrate increased
G
i2 expression in K562 cells via Sp1-binding to DNA (18). Fig. 2A shows a 6.4-fold
increase in the endogenous expression of G
i2 when 2 mM valproic acid was used; this -fold increase is similar
to that shown in Fig. 1B at this concentration of valproate. As judged by the decreased intensity of G
i2 signal on
Western blots (Fig. 2A), treatment with mithramycin A not
only decreased valproate-induced expression of the endogenous
G
i2 gene (compare lane 3 with lane
2) but also the butyrate-induced effect (compare lane 5 with lane 4). By itself, mithramycin A had no effect on basal G
i2 expression. In transfection experiments with
pG
i2(
1214/+115)-luc, mithramycin A drastically
inhibited (87%) valproate-induced as well as butyrate-induced
G
i2 gene promoter activity (Fig. 2B). The
results of these experiments with mithramycin A suggest the involvement
of Sp1-mediated gene transcription in the action of valproic acid in
the expression of G
i2.

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Fig. 2.
Mithramycin A inhibits valproate- and
butyrate-induced G i2 gene
expression. K562 cells (1 × 105 cells/well in 1 ml of medium), grown in 24-well plates for 24 h, were transfected
with pG i2( 1214/+115)-luc, as described under
"Experimental Procedures." Mithramycin A (100 nM) was
added to the cells 1 h after transfection with the reporter
plasmid, followed by the addition of sodium valproate (2 mM) or sodium butyrate (2.5 mM) 1 h later.
The cells were harvested 24 h after the addition of valproate or
butyrate and processed for luciferase assay and Western blotting, as
described under "Experimental Procedures." A,
mithramycin A inhibits butyrate- and valproate-induced
G i2 levels. G i2 levels were determined by
Western blotting, using 10 µg of protein for each sample. The same
blot was reprobed for -tubulin as a loading control. The plot is a
densitometric quantification of three Western immunoblots similar to
that shown in the inset. B, mithramycin A
inhibits butyrate- and valproate-induced transcriptional activity.
Luciferase activities were normalized to protein content of each sample
and are expressed as -fold stimulation, relative to cells that were not
treated with valproic or butyric acid. All promoter activities were
corrected for basal activity of GL3-basic. Values shown are means ± S.E. for triplicate assays from three different experiments.
Con, control; B, sodium butyrate; Mit,
mithramycin A; V, valproic acid.
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MAPKs Are Not Involved in Valproate-induced
Transcription--
Because butyrate-induced, but not trichostatin
A-induced, transcription from the G
i2 gene promoter was
previously shown to involve the MEK-ERK signaling pathway (18), we
tested whether this pathway might be involved in the valproic acid
effect by carrying out co-transfection experiments with an expression
plasmid harboring the gene for dominant negative ERK2. Transfection
with this expression plasmid inhibited butyrate-induced transcription but had no effect on valproate-induced transcription, indicating lack
of involvement of the MEK-ERK signaling pathway in the valproic acid
effect (Fig. 3A). We also
found that, unlike butyrate (18), valproic acid-induced transcription
was not affected by U0126, a selective inhibitor of the MEK-ERK (Fig.
3B). Furthermore, selective inhibitors of p38 and c-Jun
N-terminal kinase MAPKs (i.e. SB 203580 and PD 169316) also
had no effect on valproate-induced transcription (Fig. 3B).
These data indicate that the transcriptional effect of valproate is not
influenced by any of the standard MAPK modules and therefore may be
accounted for, most likely, by its inhibition of histone
deacetylase.

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Fig. 3.
Valproic acid-induced transcription from the
G i2 gene promoter does not involve
MAPKs. A, 0.25 µg of the reporter plasmid was
co-transfected with 0.25 µg of an expression plasmid harboring the
gene for dominant negative ERK2 (dnERK2) or the empty vector
(pCEP4L) 1 h prior to the addition of sodium valproate
(2 mM) or sodium butyrate (2.5 mM). The cells
were harvested 24 h later and luciferase activity was determined
as described under "Experimental Procedures." Results shown are
means ± S.E. for duplicate samples from three separate cell
cultures. B, the MAPK inhibitors were added 1 h before
the addition of valproic acid. Results shown are means ± S.E. for
triplicate replications from three or four separate experiments. In
both A and B, promoter activity is expressed as
-fold stimulation over the appropriate controls. Con, no
addition of VPA; VPA, valproic acid; SB, SB
203580; PD, PD 169316. *, significantly different
(p < 0.05) from cells treated with butyrate
alone.
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Sp1-binding Sites Are Involved in Valproate-induced Transcription
from the G
i2 Gene Promoter--
Inhibition of histone
deacetylase, a transcriptional repressor, can release an inhibitory
constraint on Sp1 (22), thereby allowing this transcription factor to
influence transcription. To further test the idea that Sp1 is involved
in valproic acid-induced transcription from the G
i2 gene
promoter, truncation mutants of the G
i2 gene promoter
(18) were used in transient transfection assays. We found that valproic
acid-induced promoter activity was unaffected when deletions occurred
in the
1214/
184 region (data not shown), a region that contains
four of the seven putative Sp1-binding sites in the human
G
i2 gene promoter (15-17). However, when the truncation
was extended to
79 bp, involving only one (
92/
85) of the
remaining three Sp1-binding sites, we observed a 30-35% depression of
transcription. Therefore, several promoter constructs that were mutated
at these sites were used in further transfection experiments, as
indicated in Fig. 4. In this figure, the
letters M and D are used to designate point
mutations (M) and sequence deletions (D),
respectively. Single point mutations at sites 1 (+68/+75), 2 (
50/
36), and 3 (
92/
85) inhibited transcription by 34, 21, and 36%, respectively, whereas deletion of Sp1 sequences at sites 2 and 3 (mutants pD2 and pD3 in Fig. 4)
inhibited transcription by 42 and 39%, respectively. Double sequence
and point mutations at sites 2 and 3 (mutants pD2,3 and
pM2,3 in Fig. 4) resulted in 59 and 46% inhibition of
transcription, respectively. When all three sites were mutated
(i.e. triple mutant, pM1,2,3), transcription was
inhibited by 55-60% (Fig. 4), indicating that all three putative Sp1
sites (
92/
85,
50/
36, and +68/+75) contribute to the
transcriptional response to valproic acid.

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Fig. 4.
Valproate-induced promoter activities of
wild-type and mutant G i2 gene
promoters. The different mutant promoters used here have been
previously described (18). The underlined nucleotides in the
promoter maps represent substitution mutations. Nucleotide sequence
deletions are indicated with zig-zag lines within the maps.
Transfection assays were carried out as described under "Experimental
Procedures." Sodium valproate (2 mM) was added 1 h
after transfection with the reporter plasmid, and the cells were
harvested for luciferase assay 24 h later. Values shown are
means ± S.E. for triplicate assays from four different
experiments. The luciferase activities of the cell extracts are
expressed as -fold stimulation, relative to cells that were not treated
with valproic acid. Nucleotide substitutions (M series) and
sequence deletions (D series) of putative Sp1 motifs at
positions +68/+75, 50/ 36, and 92/ 85 are indicated throughout as
Sp1 sites 1, 2, and 3, respectively. Statistical analysis is with
reference to the wild-type ( 1214/+115) promoter. *, p < 0.01; #, p < 0.05. LUC, luciferase
reporter gene.
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Binding of Sp Family of Transcription Factors to DNA
Probes--
Electrophoretic mobility shift assays were performed with
three different labeled double-stranded DNA probes containing
corresponding nucleotide sequences at locations 1 (+68/+75), 2 (
50/
36), and 3 (
92/
85) in the G
i2 gene promoter.
For each DNA probe, the results showed three binding complexes that are
typical of nuclear extract binding to Sp1/Sp3-binding element (28);
therefore, only one such EMSA is shown (Fig.
5). A consensus Sp1 oligonucleotide completely abolished the binding of nuclear proteins to the three DNA
probes (Fig. 5, lane 3). Corresponding unlabeled Sp1
oligonucleotides also abrogated binding to these probes (lane
5), whereas a mutated Sp1 oligonucleotide (lane 4) and
an unrelated (NF-
B) oligonucleotide (lane 6) had no
effect. As expected, mithramycin A interfered with this binding
(lane 15).

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Fig. 5.
EMSA. K562 cells (2 × 106 cells) were grown for 24 h in 20 ml of medium,
using 100-mm Petri dishes. Valproic acid (2 mM) was then
added to some dishes, and the cells were harvested after 24 h.
Nuclear extracts were prepared as previously described (18). Annealed
oligonucleotides, used as probes, were labeled with
[ -32P]dCTP by using the Klenow fill-in reaction. Using
nuclear extracts from control and valproic acid-treated cells, EMSA was
performed as described in detail elsewhere (18). The reactions were
carried out with 2 µg of nuclear extract protein for each lane.
Competition experiments were carried out with a 50-fold excess (or
more) of Sp1 consensus oligonucleotide (lane 3),
mutated unlabeled oligonucleotide (lane 4),
unlabeled oligonucleotide identical (in sequence) to the labeled
oligonucleotide (lane 5), and an unrelated (NF- B)
oligonucleotide (lane 6). Mithramycin A (final
concentration = 5 × 10 4 M) was
incubated with the labeled probe for 30 min at 25 °C before adding
nuclear extract (lane 15). The anti-Sp1 antibodies used in
lanes 7 and 8 were from Santa Cruz Biotechnology
and Upstate Biotechnology, respectively. All other antibodies were from
Santa Cruz Biotechnology. The EMSA shown was performed with the probe
5'-GTGGGTCGGGCGGGGCCGAGCCG-3', which contains the putative
Sp1-binding site (underlined) designated as site 1 throughout. EMSAs
were also performed with 5'-ACCCCCGGCCCGCCCCGCCGTCG-3' and
5'-GCCTGCAAGCCCGCCCCGGCCCAGTCACA-3', which contain the
putative Sp1-binding sites (underlined) designated as sites 2 and 3, respectively, throughout. With all three probes, the EMSA patterns were
similar. NE, nuclear extract; Ab, antibody;
oligo, oligonucleotide.
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To determine the protein composition of these complexes, antibodies to
members of the Sp family of transcription factors were added to the
DNA-binding assay. For all three DNA probes, supershifted protein-DNA
complexes were observed with antibodies to Sp1, Sp2, and Sp3, not only
in nuclear extracts from control cells (Fig. 5, lanes 7-10)
but also in nuclear extracts from cells treated with valproic acid
(lanes 12-14). These results confirm that these three sites
(
92/
85,
50/
36, and +68/+75) in the human G
i2 gene promoter bind to members of the Sp family of transcription factors. Although Sp2 is not known to recognize the same sequence (GC-box) as Sp1 or Sp3 (27-29), it has been reported by others to bind
to DNA probes designed to detect Sp1/Sp3-binding sites (30). This
binding is probably attributable to the flanking GT-containing sequences in the probes used. Taken together with the
data in Fig. 4, we conclude that these Sp1/Sp3-binding sites are
relevant to the valproic acid-induced transcription from the G
i2 gene promoter. Given that mutations in these
critical Sp1/Sp3-binding sites did not completely suppress valproic
acid-induced transcription (Fig. 4), the possibility that other
transcription factor(s) may also be involved in the valproic acid
effect cannot be ruled out.
Dephosphorylating Conditions Enhance Sp1/Sp3 Binding to
DNA as Well as Valproic Acid-induced Transcription from the
G
i2 Gene Promoter--
In Fig. 5, it is clear that the
intensity of the protein-DNA signal was greater in cells treated with
valproic acid than in control cells (lane 11 versus lane 2; also compare lanes
12-14 with lanes 7-10). This might suggest either an
increased affinity of nuclear proteins for the labeled DNA or an
increased nuclear content of these transcription factors in the
valproate-treated cells. We did not detect any change in the nuclear
content of Sp1, Sp2, or Sp3, as measured by Western blotting of nuclear
extract samples (Fig. 6). Because
transcriptional action of Sp1 can be influenced by its phosphorylation
state (31-33), we reasoned that the increased signal intensity
probably resulted from post-translational modification of the
transcription factor(s).

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Fig. 6.
Valproic acid has no effect on the expression
of Sp family of proteins. Nuclear extracts (10 µg of protein)
from valproic acid-treated and control cells were analyzed by
immunoblotting for Sp1 (A), Sp2 (B), and Sp3
(C) protein content. The nuclear extracts used for these
Western blots were the same as those used for EMSAs in Fig. 5.
The blots were reprobed for C/EBP (D) and c-Fos
(E) as loading controls. The blots shown are representative
of results obtained with at least two separate preparations of nuclear
extracts. VPA, valproic acid.
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Protein phosphatase had previously been shown to dephosphorylate Sp1
and thereby increase the binding of this transcription factor to GC
boxes on DNA (31, 32, 34). Also, alkaline phosphatase treatment of
nuclear extracts has been shown to dephosphorylate Sp1 in
vitro (35). As shown in electrophoretic mobility shift assays
(Fig. 7), alkaline phosphatase treatment
of nuclear extracts increased binding of Sp proteins to the same DNA
probes used in Fig. 5. Nonspecific bands were not affected by this
treatment. If this increased binding is relevant to the transcriptional
activity of valproate, then a decrease in valproate-induced
transcription should be expected when transfection experiments are
carried out under conditions that would engender dephosphorylation
in situ. Therefore, we used calyculin A and okadaic acid,
two potent inhibitors of protein phosphatase 1 (PP1) and 2A (PP2A) (36,
37) to inhibit protein phosphatase and then measured the effect of this
inhibition on valproate-induced promoter activity. The results show
that these compounds potently inhibited valproic acid-induced
transcription in a concentration-dependent manner;
furthermore, fostriecin, another protein phosphatase inhibitor that
exhibits much greater inhibitory potency against PP2A than PP1 (38),
also inhibited valproic-acid induced transcription (Fig.
8A). The protein phosphatase inhibitors were effective only against the wild-type promoter but not
against promoter constructs in which Sp1-binding sites had been mutated
(Fig. 8B). These results are consistent with the idea that
modulation of the phosphorylation status of Sp family of transcription
factors is a mechanism by which valproic acid induces
G
i2 gene expression. This idea was further supported by
electrophoretic mobility shift and supershift assays using nuclear
extracts from cells treated with valproic acid and okadaic acid (Fig.
9). In these assays, the binding of
nuclear extract proteins from cells treated with valproic acid and
okadaic acid (Fig. 9, lanes 11-13) was significantly
attenuated compared with nuclear extracts from cells that were treated
with valproic acid alone (lanes 8-10). At the concentration
used, okadaic acid, by itself, had no effect on nuclear extract binding
to the probes used (Fig. 9, compare lanes 5-7 with
lanes 2-4). Taken together with the data in Figs. 7 and 8,
these results support the idea that dephosphorylating conditions
enhanced Sp1/Sp3 binding to DNA as well as transcriptional activity of
these transcription factors.

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Fig. 7.
Alkaline phosphatase treatment of nuclear
extracts increases binding to Sp1-binding DNA probes. Nuclear
extracts (1 µg of protein) from K562 cells were incubated in buffer
with calf intestinal alkaline phosphatase for 20 min at 37 °C. The
amounts of enzyme used are indicated as units of enzyme activity. At
the end of the 20 min, annealed oligonucleotide labeled with
[ -32P]dCTP was added to the tubes, and the mixture was
used for the gel mobility shift assay. The data shown are
representative of three such experiments, using two different DNA
probes identical to those used in Fig. 5. Alk. Phos.,
alkaline phosphatase; NE, nuclear extract.
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Fig. 8.
Effect of protein phosphatase inhibitors on
valproate-induced promoter activities of wild-type and mutant
G i2 gene promoters. K562
cells (1 × 105 cells/well in 1 ml of medium), grown
in 24-well plates, were transfected with wild-type or mutant
G i2 gene promoters, as described under "Experimental
Procedures." The mutant promoters contained mutations in Sp1-binding
sites and were described in Fig. 4. The protein phosphatase inhibitors
were added to the cultures 1 h before the addition of valproic
acid (2 mM), and the cells were harvested for luciferase
assay 24 h later. For each inhibitor concentration, promoter
activity in the absence of valproic acid was equated to 1.0 and used as
control to calculate -fold stimulation in the presence of valproic
acid. Values shown are means ± S.E. for triplicate assays for
four different cell cultures in A and three in B.
All values are corrected for basal activity of GL3-basic. A,
dose-dependent effect of three protein phosphatase
inhibitors (okadaic acid, calyculin A, and fostriecin) on the activity
of the wild-type G i2 gene promoter
(pG i2( 1214/+115)-luc). B, lack of effect of
the protein phosphatase inhibitors on mutant G i2 gene
promoters. Con, control (no VPA added); VPA,
valproic acid; WT, wild type; pD2,3,
mutant promoter construct in which Sp1-binding motifs at sites 2 and 3 were deleted (see Fig. 4); pM1,2,3,
mutant promoter construct containing nucleotide substitution mutations
at Sp1-binding sites 1, 2, and 3, as shown in Fig. 4.
|
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|
Fig. 9.
Treatment of K562 cells with okadaic acid
decreases binding of Sp1/Sp3 to DNA. K562 cells (2 × 106 cells) were grown for 24 h in 20 ml of medium,
using 100-mm Petri dishes. In cells that were treated with valproic
acid and okadaic acid, okadaic acid (20 nM) was added
1 h before the addition of valproic acid (2 mM). The
cells were harvested 24 h later, and nuclear extracts were
prepared. EMSA was performed as described in detail in the legend to
Fig. 5, using DNA probes specific to binding sites designated 1 and 2 in the legends to Fig. 5 (see "Results"). The results with
both probes were similar. The representative figure shown here was
generated with the probe (5'-GTGGGTCGGGCGGGGCCGAGCCG-3'),
which contains the putative Sp1-binding site (underlined),
designated as site 1. All reactions were carried out with 2 µg of
nuclear extract protein. Supershift assays were carried out with 2 µg
of each antibody used. Control, nuclear extracts from cells
that were not treated with either valproic acid or okadaic acid;
Ab, antibody; VPA, valproic acid; OA,
okadaic acid; NE, nuclear extract.
|
|
 |
DISCUSSION |
Valproic acid is the active ingredient in divalproex sodium
(depakote) that is used clinically in the treatment of epilepsy. In
addition to its anticonvulsant activity, valproic acid also exhibits
teratogenic effects in humans and animals (9, 39-44). The mechanism by
which valproic acid brings about these effects is not known. A recent
study involving induction of differentiation in F9 embryocarcinoma
cells has suggested that valproic acid induces the expression of
peroxisome proliferating activating receptor
and has associated
this induction with the occurrence of valproic acid-induced
teratogenicity (45). Valproic acid-induced gene expression (8-11)
has been attributed to the DNA-binding activity of the transcription
factor AP-1. However, it is unclear whether the gene
expression-inducing effect of valproic acid can be attributed solely to
its activation of AP-1 binding to DNA. In fact, Phiel et al.
(9) had noted that valproic acid robustly activated transcription from
Renilla luciferase reporter gene driven by the
cytomegalovirus promoter (pRL-CMV), which they indicated does not
contain AP-1 sites.
In the present study, we have used the human G
i2 gene
promoter, which does not contain AP-1 sequence motifs (15-17), to
illustrate involvement of the Sp family of transcription factors in the
transcriptional activity of valproic acid. Our results show that in
K562 cells, valproic acid-induced transcription from this promoter was
inhibited by mithramycin A, a potent inhibitor of Sp1 binding to GC
boxes in DNA (23-27). Valproic acid-induced transcription was also
substantially depressed when three Sp1-binding sequences in the human
G
i2 gene promoter were mutated. Electrophoretic mobility
shift assays indicated that these sequences bind not only Sp1 but also
Sp2 and Sp3, three closely related members of the Sp family of
transcription factors. Together with the results of our reporter gene
assays, these data support the conclusion that one or more members of
the Sp family of transcription factors is involved in the
transcriptional effect of valproic acid on the human G
i2
gene promoter. This study provides a substantially different
perspective from reports (8, 13) that defined the AP-1 response element
as the modality by which valproic acid induces gene expression. The
finding that mutations at these critical Sp1 sites from the
G
i2 gene promoter did not completely suppress
transcription in our cellular transfection system suggests that other
transcription factor(s) besides Sp family members may also be involved
in the demonstrated transcriptional effect of valproic acid.
Histone deacetylase (HDAC) inhibitors are increasingly gaining
attention in cancer research (46-50) because of their ability to
inhibit cell proliferation. Valproic acid is a new addition to this
list and was shown recently to inhibit histone deacetylase in
Xenopus and human embryonic kidney (293T) cells (9).
Göttlicher et al. (49) also demonstrated that valproic
acid is a powerful inhibitor of HDAC, relieves
HDAC-dependent transcriptional repression, and causes
hyperacetylation of histones in vivo and in cultured cells.
Taken together with the studies of Doetzlhofer et al. (22) that show that inhibition of HDAC by HDAC inhibitors releases an
inhibitory constraint on Sp1, making it possible for this transcription factor to associate with other accessory proteins to effect
transcription, our present study provides an important dimension to
further understanding of mechanisms underlying valproic
acid-induced gene transcription.
Valproic acid-induced transcription demonstrated in this study was
sensitive to three powerful inhibitors of PP1 and PP2A (i.e.
okadaic acid, calyculin A, and fostriecin), indicating that it requires
the action of protein phosphatase. These chemical inhibitor studies,
however, do not precisely pinpoint which protein phosphatase may be
involved. That the transcriptional action of Sp1 can be influenced by
its phosphorylation state has been amply demonstrated (31-33, 52, 53).
In this context, it should be noted further that Lacroix et
al. (34) recently demonstrated, in interleukin-2-treated T
lymphoma cell line Kit225, that PP2A but not PP1 dephosphorylates Sp1,
resulting in increased binding of Sp1 to DNA as well as up-regulation
of its transcriptional activity with respect to the human
immunodeficiency virus type 1 long terminal repeat promoter as well as
a chloramphenicol acetyltransferase reporter gene under the control of
six tandem Sp1-binding sites from SV40 early promoter (SV40-tkCAT).
Also, Li et al. (54) recently showed in studies with mouse
embryonal carcinoma P19 cells that retinoic acid-induced transcription
from the mouse
-opioid receptor gene promoter is mediated by
increased binding of dephosphorylated Sp1 to GC box elements in that
promoter. We can infer from their studies and from our protein
phosphatase experiments (Figs. 7-9) that dephosphorylated Sp1/Sp3
mediated valproic acid-induced transcription from the
G
i2 gene promoter observed in our present study. Besides
Sp1, not much is known about post-translational modification(s) that
might influence the transcriptional activity of Sp family of
transcription factors. Our present work suggests that, like Sp1,
phosphorylation state of Sp3 might influence its DNA
binding/transcriptional activity. Interestingly, two research groups
(55, 56) showed recently that another post-translational modification
(i.e. sumoylation) influences whether Sp3 functions as a
repressor or activator of transcription.
 |
ACKNOWLEDGEMENTS |
We thank Jianqi Yang for very valuable
discussions during the performance of this work and for assistance in
the electronic preparation of the figures in the manuscript. Dr. Lee
Weinstein kindly provided the plasmid
pG
i2(
1214/+115)-CAT containing wild-type promoter for
the human G
i2 gene. We are also grateful to Dr. Melanie
Cobb for providing us with the expression plasmid harboring the gene
for dominant negative ERK2.
 |
FOOTNOTES |
*
This work was supported by National Science Foundation Grant
MCB-9905070.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry,
Meharry Medical College, 1005 Dr. David B. Todd, Jr., Blvd., Nashville,
TN 37208-3599. Tel.: 615-327-6586; Fax: 615-327-6442; E-mail:
iarinze@mail.mmc.edu.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M209430200
 |
ABBREVIATIONS |
The abbreviations used are:
AP-1, activator
protein 1;
C/EBP
,
-isoform of CCAAT box enhancer-binding protein;
G
i2, isoform 2 of the
-subunit of inhibitory guanine
nucleotide-binding regulatory protein;
EMSA, electrophoretic mobility
shift assay;
ERK, extracellular signal-regulated protein kinase;
HDAC, histone deacetylase;
MAPK, mitogen-activated protein kinase;
MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase
kinase;
PP1 and PP2A, protein phosphatase isoform 1 and 2A,
respectively;
Sp1, Sp2, and Sp3, promoter-specific factor-binding
proteins 1, 2, and 3 (also called specificity proteins 1, 2, and 3).
 |
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