Laboratoire de Biologie Moléculaire et de Génie
Génétique (I.M., J.A.M., M.M.) Université de
Liège Institut de Chimie B6 B-4000 Sart-Tilman,
Belgium
Laboratoire dimmunopathologie (L.C.) INSERM
U430 Hopital Broussais 75014 Paris, France
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ABSTRACT |
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INTRODUCTION |
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In addition, although less exhaustively studied, PRL gene expression was shown to be regulated by protein phosphatases (12, 13). Protein phosphatases are involved in the interruption of signal transduction by reverting phosphorylation events on defined target proteins. Integration of external signals in a cell is partially based on the equilibrium between the activities of protein kinases and phosphatases (14, 15, 16). Thus, understanding regulation of gene transcription in a cell would not be complete without studying the effects of protein phosphatases. The growing numbers of protein phosphatases are classified according to their specificity as serine/threonine phosphatases, tyrosine phosphatases, or dual specificity phosphatases. About half of them are serine/threonine phosphatases, four major types (PP1, PP2A, PP2B, and PP2C) of which have been genetically defined (17). The Ca2+-dependent protein phosphatase, PP2B, and okadaic acid (OA)-sensitive phosphatases, such as PP1 or PP2A, regulate hPRL gene transcription via the proximal region of the promoter (12, 13).
The tumor promoter OA inhibits the two major intracellular protein phosphatases, PP1 and PP2A; the in vitro inhibition of PP2A occurs at nanomolar concentrations and that of PP1 at micromolar doses (18). Recently, it was shown that low concentrations of OA also inhibit other serine/threonine protein phosphatases including PP3, PP4, and PP5 (19, 20, 21). Despite this limitation, the use of OA has provided useful information on the implication of protein phosphatases in many cellular regulations. In rat pituitary GC cells, low OA concentrations (30 nM) stimulate the basal activity and potentialize the cAMP stimulation of the proximal PRL promoter region, linked to the thymidine kinase (TK) promoter. Both effects were shown to involve sequence A (12).
The aim of our work was to further analyze the effect of protein phosphatases on the hPRL proximal promoter. We investigated the OA regulation of the natural hPRL promoter activity in GH3B6 cells that express the endogenous PRL gene. OA stimulation was studied at the level of transcription factor binding, using nuclear extracts from OA-treated and untreated cells, and at the functional level, using transient transfection experiments. Our results show that OA induces binding of JunD and c-fos to a newly identified AP1 motif within sequence P1 of the hPRL proximal promoter and in parallel increases JunD and c-fos cellular content. Moreover, we demonstrate that basal activity and OA stimulation of the hPRL proximal promoter require this AP1 site. Although both AP1 and Pit-1 are needed to obtain OA stimulation of the hPRL proximal promoter activity, the binding of each to sequence P1 is independent.
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RESULTS |
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Transient transfection experiments were performed using a reporter
plasmid containing the luciferase gene under the control of the
proximal 164 bp of the pituitary hPRL promoter (164P-Luc). Within this
part of the promoter (Fig. 1A), footprint
experiments have defined two protected regions (P1 and P2) containing
Pit-1 binding sites (4). In addition, gel retardation assays have
revealed binding of a ubiquitous protein to sequence A (5). Moreover,
the 164- bp region is sufficient to mediate the response to almost all
second messengers and is the target for phosphatase inhibitor effects
(12). OA stimulation of this construct was analyzed in GH3B6 cells and
compared with a control construct containing only 40 bp upstream of the
hPRL start site (40P-Luc), which comprise only the TATA box. In this
system, OA stimulates transcription of the 164P-Luc construct
dose-dependently with the maximal stimulation (45-fold) when cells are
treated with 80 nM of OA (Fig. 1B
). At concentrations lower
than 20 nM, we observed a similar OA stimulation of both
the 164P-Luc and the 40P-Luc. These effects were considered as
nonspecific and may result from OA action on the basal transcription
machinery, as previously discussed by Wera et al. (12). At
higher concentrations, stimulation of the 164P-Luc was much stronger
than stimulation of the control, which reflects the OA action on the
hPRL promoter.
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Appearance of a C4 complex on the P1 sequence is observed in some
experiments. This complex is displaced by oligonucleotides P1, P2, and
by an oligonucleotide containing the consensus binding site for Oct-1.
Oct-1 is a transcription factor related to Pit-1 and able to bind
similar sequences (Fig. 2B, lane 6). Thus, this C4 complex corresponds
to a protein related to these transcription factors. The C4 complex is
not induced by OA treatment, as it is observed in treated and untreated
extracts (data not shown). Furthermore, the C3 complex is not affected
by oligonucleotides that inhibit C4 binding, clearly indicating that
the two complexes are distinct. Thus, the C4 complex was not
investigated further.
Binding of the C3 complex is not affected by an excess of
oligonucleotide P2 (Fig. 2B lane 3), again indicating that it does not
correspond to the binding of Pit-1. Similarly, complex C3 is not
displaced by an unlabeled oligonucleotide containing a binding site for
Oct-1, whereas this oligonucleotide displaces both C1 and C2 complexes
(lane 6). An oligonucleotide containing a consensus site for the
transcription factor NFY, used as a nonspecific competitor, does not
displace any of the complexes (lane 7). These observations show that
complexes C1, C2, and C4 contain Pit-1 or a related protein, while C3
clearly involves a different transcription factor.
A careful analysis of the P1 sequence revealed a degenerated putative
binding site for cAMP response element-binding protein (CREB) or AP1
transcription factors (Fig. 1A). Therefore, competition experiments
were performed using DNA fragments containing a consensus site for
these two factors. Both unlabeled oligonucleotides displace the C3
complex from P1 specifically (Fig. 2B
, lanes 4 and 5), without
affecting Pit-1 binding (monomeric or dimeric form). Moreover, a
complex migrating as the C3 complex is observed when gel retardation
assays are performed using labeled oligonucleotides containing CREB or
AP1 consensus sequences (data not shown). These data strongly suggest
that this additional C3 complex corresponds to CREB or AP1.
Specific antibodies were then used to further investigate the nature of
the factors involved. Antibodies against Pit-1 strongly affected
complexes C1 and C2, both in untreated (Fig. 3A, compare lanes 2 and 3) or OA-treated
cells (compare lanes 5 and 6), confirming that they consist of Pit-1.
The supershift in this case migrates at a position similar to complex
C3 (lane 3). A sharp band with a very low mobility was also present
when Pit-1 antibody was used alone (lane 11). Anti-CREB antibodies
directed against the entire protein (anti-CREB 253) or against the
kinase-inducible domain (anti-CREB 244) did not alter formation of the
C3 complex (lanes 8 and 9). However, they were able to inhibit binding
of CREB to a consensus cAMP response element (CRE)-binding site (data
not shown). Thus, complex C3 does not contain CREB. On the contrary,
antibodies directed against the DNA-binding domain of c-jun
inhibit formation of the C3 complex, indicating that it corresponds to
AP1 (Fig. 3A
, lane 7). As these antibodies recognize c-jun,
JunB, and JunD, more specific antibodies directed against the more
variable C-terminal region of these proteins were used to determine the
precise nature of the C3 complex. Figure 3B
shows that only anti-JunD
antibody is able to form a supershifted complex (lane 3). Specific
antibodies directed against members of the fos family were used to
demonstrate that a supershift was obtained using anti-c-fos
antibody, but not using anti-fosB antibody (Fig. 3C
, lanes 2 and
3). Appearance of both the junD and c-fos supershift
correlates with a slight decrease of complex C3 formation. Complete
disappearance of complex C3 was not obtained, due to the low
concentration of the antibodies used in these experiments. The
supershifts were not obtained using the antibodies alone (Fig. 3B
and
C, lane 4) or using untreated extracts, further showing that they are
specific and that they arise from complex C3. Thus, OA induces binding
of an AP1-related complex, containing JunD and c-fos, on the
P1 site of the hPRL promoter.
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Western blot experiments were performed to determine whether
modification of the binding on the P1 site reflects OA regulation of
the cellular concentration of Pit-1, JunD, or c-fos
proteins. Figure 4C shows that the cellular amounts of JunD and
c-fos proteins increase upon OA treatment of the cells.
These increases are dose-dependent and could account for the OA
induction of AP1 binding to P1 sequence and formation of the
C3 complex. Western blot experiments reveal a decrease of the Pit-1
binding after OA treatment (Fig. 4C
), correlating with the decrease of
the dimer complex in gel retardation experiments (Fig. 4B
). This
decrease could be due to a long-term decrease of Pit-1 content, which
has been previously described (22, 23). Nevertheless it should be noted
that the C2 complex is much more affected by OA treatment than C1,
which could also reflect the lower binding affinity of C2 on site P1
(24).
OA Stimulation of the 164P-Luc Construct Is Mediated by
Pit-1 and AP1 Binding Sequences
To study the functional implication of AP1-like
factors in the OA induction of the hPRL gene, different reporter
constructs, containing the first 164 bp of the hPRL promoter, were
transiently transfected in GH3B6 cells. Wild-type 164P-Luc and
constructions mutated within the P1 site, either in the AP1 or
in the Pit-1 consensus sequence (Fig. 5A), were transfected in parallel to
determine the respective role of each transcription factor. The
abolition of the respective Pit-1 or AP-1 binding was verified using
gel retardation assays. A P1 oligonucleotide mutated in the Pit-1
motif showed that this mutation inhibits binding of Pit-1 (Fig. 5B
, lanes 5 and 6) without affecting AP1 binding. Conversely, a P1
oligonucleotide mutated in the AP1 motif does not bind AP1 and is still
able to bind Pit-1 (Fig. 5B
, lanes 8 and 9).
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Basal activity of the 164P-Luc construct (3750 ± 847) is reduced 3-fold when either the AP1 (164AP1 m-Luc) or the Pit-1 (164Pit-1 m-Luc) site is mutated (1141 ± 335 and 1212 ± 122, respectively), but remains higher than the activity of 40P-Luc (449 ± 22). This indicates that both transcription factors are required for the basal transcription of the hPRL promoter. Moreover, both mutations lead to a similar reduction of basal activity, suggesting that AP1 is as important as Pit-1 in basal hPRL gene expression.
To analyze OA stimulation, the fold induction at each OA concentration
was represented in Fig. 5C and basal values at 0 nM OA were
set to 1 for each curve. OA stimulation of the 164P-Luc construction is
abolished when either the Pit-1 or the AP1 consensus sites are mutated.
Both mutated constructs respond to OA stimulation to the same level as
the 40P-Luc (Fig. 5C
) at each OA concentration tested.
In conclusion, both Pit-1 and AP1 sites are required for an efficient stimulation of the hPRL proximal promoter by OA.
AP1 and Pit-1 Bind to Sequence P1 Independently
The functional importance of both Pit-1 and AP1 in OA stimulation
prompted us to investigate more precisely their interactions with
sequence P1. First, we used specific antibodies to analyze the
complexes formed and to determine whether the two transcription factors
associate to form a heterodimer. The anti-Pit-1 antibody is directed
against the N-terminal part of Pit-1 and displaces C1 and C2 complexes,
which both correspond to Pit-1 binding (Fig. 3A). Unfortunately, the
supershift obtained comigrates with the C3 complex, thus preventing a
conclusion about the presence of Pit-1 in the C3 complex. To circumvent
this problem, gel retardation assays were performed using unlabeled P1
oligonucleotide and blotted on a nylon membrane. The complexes obtained
were revealed using specific anti-Pit-1 or anti-c-jun
antibodies (Fig. 6
). Anti-Pit-1 antibody
is able to detect complexes C1 and C2, but most importantly does not
recognize the C3 complex (lanes 3 and 4). On the contrary,
anti-c-jun antibody specifically detects complex C3, but not
C1 or C2 (lanes 5 and 6). Most of the anti-c-jun
immunoreactive compounds remained at the top of the gel, probably
reflecting the lower affinity of sequence P1 for AP-1. Note also that
the c-jun antibody binds weakly to the jun-DNA complexes,
since it is directed against the DNA-binding domain. We conclude that
the C3 complex contains jun-related factors, but no
significant amounts of Pit-1, suggesting that C3 does not consist of an
AP-1/Pit-1 heterocomplex.
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DISCUSSION |
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We analyzed OA regulation of transcription factor binding to the hPRL
proximal promoter [-164/+1], shown to be sufficient for OA
stimulation (Ref. 12 and Fig. 1B). Gel retardation experiments using
sequences P1 and P2, previously known to bind Pit-1, show that OA
treatment induces the appearance of an additional complex (C3)
specifically on sequence P1, but not on P2. Supershift experiments show
that only anti-junD and anti-c-fos antibodies bind to
complex C3 (Fig. 3
); competition experiments show that Pit-1-specific
oligonucleotides (P2 or P1,AP1 m) (Figs. 2
and 7
) or mutation of the
Pit-1 site (Fig. 5B
) do not affect formation of the complex C3,
strongly suggesting that the OA-induced P1 complex is composed of the
AP1-related transcription factors JunD and c-fos.
The functional implication of AP1 in OA stimulation is supported by
several observations. First, we have observed an increase of AP1
binding to a consensus AP1 binding site and to P1 (Fig. 4, A and B),
which perfectly correlates with the stimulation of the hPRL promoter
(Fig. 1
). Second, a parallel, clear and specific increase in JunD and
c-fos protein is observed in Western blot analysis. Finally,
mutation of the AP1 binding site in sequence P1 reduces OA stimulation
to the level of the minimal promoter 40P-Luc (Fig. 5C
).
The c-jun and c-fos protooncogenes were originally identified as components of the AP1 transcription complex. Through protein-protein interactions within the leucine zipper domain, c-jun can form either homodimers or heterodimers with other leucine zipper-containing proteins, such as other Jun family proteins (JunB, JunD), members of the Fos (c-fos, fosB, fra1, fra2), or members of the CREB/activation transcription factor (ATF) families. Phosphorylation has been implicated in the regulation of AP1 function. Phosphorylation of residues located in the C-terminal domain of c-jun, by the CKII and the GSK-3, inhibits its DNA-binding activity (25, 26). Conversely, phosphorylation of serine residues located in the N-terminal part of c-jun by Ha-ras (27, 28) or JNK (29) leads to the increase of c-jun transactivating properties. Although these residues are well conserved in JunD, it has been recently shown that both hypophosphorylated or hyperphosphorylated forms of JunD are able to bind DNA (30). Thus, phosphorylation at the C-terminal sites of junD may not account for the increase in binding to the P1 site observed in OA-treated cells. Although we cannot completely rule out an increase of the AP1 transactivation function by phosphorylation of the N-terminal region, OA stimulation of the hPRL promoter activity appears to result from up-regulation of AP1 expression.
In other systems, two complementary mechanisms have been shown to lead
to accumulation of c-jun and could also account for JunD
accumulation. First, a positive feedback by activated c-jun
leading to enhancement of c-jun gene expression, through an
AP1 site located in the c-jun promoter, has been reported (31, 32).
Moreover, in human T-cell derived Jurkat cells, OA treatment increases
AP1 binding to its response element and is associated with an increase
in mRNA levels of c-jun, JunB, and JunD (33). Thus,
increasing jun phosphorylation after OA treatment could result in
positive autoregulation of JunD expression, as has been described in
mouse keratinocytes (34). Second, an increase in protein stability by
phosphorylation could also lead to junD accumulation, such as has been
postulated in the OA induction of the urokinase-type plasminogen
activator gene by c-jun (35). However, involvement of such
mechanisms remains to be established, but they may lead to the clear
increase of jun-like factors observed in Western blot
experiments (Fig. 4C) and could account for OA stimulation of the hPRL
promoter activity.
The c-fos accumulation (Fig. 4C) may be due to activation of
the independent ERK1/2 pathway (36) by inhibition of protein
phosphatases. In addition, carboxy-terminal phosphorylation of ser362
and 374 also increases c-fos transactivating properties (37)
and may contribute to AP1 stimulation of the hPRL promoter activity in
OA-treated cells. However, c-fos is a labile protein, and the
hyperphosphorylated form appears to be unstable (38). Thus,
accumulation of c-fos after OA treatment rather reflects an
increase in c-fos expression.
OA stimulation of the hPRL proximal promoter not only involves AP1, but
also requires binding of Pit-1 to sequence P1. Our results (Fig. 5)
clearly show that mutation of the Pit-1 site in sequence P1 reduces OA
stimulation to the same extent as mutation of the AP1 motif. In
contrast to the increase of AP1 binding to sequence P1, a decrease of
Pit-1 complex formation is observed upon OA treatment, especially
considering the dimeric form. As competition experiments show that both
transcription factors bind independently to the P1 site, this decrease
could be due to the decrease in Pit-1 protein amounts observed in
Western blot experiments, which would mainly affect the homodimeric
complex due to its lower affinity (Fig. 4C
).
Recently, a decrease in protein stability was shown after hyperphosphorylation of Pit-1 after activin treatment of somatotropic cells (22). Furthermore, a 2-fold repression of the human Pit-1 promoter by AP1 was shown in GH3 cells (23). Thus, the increase of Pit-1 phosphorylation and the increase in AP1 activity upon OA treatment might result in a long-term decrease in Pit-1 protein amounts, leading to the observed decrease in dimer formation. Such a secondary decrease in Pit-1 amounts would most likely result in a decrease in hPRL transcription and cannot account for the observed involvement of Pit-1 in OA induction. Therefore, we can conclude that the mechanism for Pit-1 stimulation of the hPRL gene by OA does not occur via regulation of Pit-1 binding activity but rather relies on the modulation of its transcriptional activity.
The fact that mutation of the AP1 or the Pit-1 motif both resulted in
the abolition of the OA induction suggests a cooperative action of
these two factors. Pit-1 and AP1 do not associate to form a
heterodimer, as complex C3 (containing AP1) is not recognized by
Pit-1-specific antibody (Fig. 6), and the same complex is obtained in
gel retardation experiments using nuclear extracts from heterologous
cells that do not express Pit-1 (data not shown). Moreover, gel
retardation experiments using mutated oligonucleotides either as a DNA
probe or competitor demonstrate that AP1 and Pit-1 binding to sequence
P1 are independent. Thus, the observed synergistic induction requires
functional interaction between Pit-1 and AP1. Such an interaction
between Pit-1 and an AP1-like transcription factor has been shown to be
involved in the hormone-regulation of the human TSHß promoter in HeLa
cells (8). Both transcription factors are required for stimulation of
the human TSHß promoter activity, but they do not cooperate to bind
to their target sequences.
Interestingly, our results show that AP1 is also involved in basal activity of the proximal hPRL promoter. Mutation of the AP1 motif, which does not alter Pit-1 binding, reduces the activity of 164P-Luc to the same extent as mutation of the Pit-1 motif, suggesting that the AP1 synergism with Pit-1 operates at the basal level as well. Moreover, cotransfection of c-jun and c-fos expression vectors in HeLa cells was shown to increase the proximal hPRL promoter activity (39). It is unclear at present whether such a synergism would require the phosphorylation of one or both of the factors involved.
Our results clearly show that mutation of the AP1 or of the Pit-1 motif in element P1 abolishes OA induction of the hPRL promoter in GH3B6 cells. A previous report suggested that OA stimulation of hPRL was mediated by sequence A without involvement of the P1 site in GC cells (12). This discrepancy could be due to the different experimental procedures used. The previous study was performed using constructs containing part of the hPRL upstream region cloned in front of the TK promoter, which by itself responds to OA. Moreover, the GC pituitary cells secrete endogenous GH, but no more endogenous PRL. In contrast, our experiments were performed using hPRL promoter constructs in PRL-producing GH3B6 cells. Thus, it is possible that regulation of PRL synthesis is different in these two cell lines, due to their different intracellular protein content (i.e. transcription factors, protein phosphatases... ).
Interestingly, AP1 regulation of the rat PRL (rPRL) proximal promoter
appears to differ from AP1 regulation of the human promoter. It was
shown that c-jun inhibits the rat PRL promoter activity in
GH4 pituitary cells (40). No AP1 binding site is found in the rat PRL
proximal promoter, and it has been shown recently that this repression
depends on the presence of the inhibitory sequence FPII, the
c-jun -domain, and the cellular context (41). In the hPRL
promoter, sequence FPII is missing, and a AP1 binding site is present
in sequence P1, resulting, in this case, in an activation by AP1. Our
results represent the first description of a differential regulation
between rat and human PRL gene transcription.
In conclusion, we propose a mechanism by which AP1 binding to sequence P1 results in synergism with Pit-1, located at the most proximal binding site, leading to an increase in hPRL basal level expression. Activation by the protein phosphatase inhibitor OA increases the cellular amount of AP1-related factors (JunD/c-fos) resulting in an increase of the synergism with Pit-1 and, ultimately, induction of the hPRL transcription.
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MATERIALS AND METHODS |
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Plasmid Constructs
The first 164 bp of the hPRL promoter were obtained after
digestion with BglII and HindIII of the
p164PRL-CAT (24). Fragments were purified on polyacrylamide gel and
then inserted in the pXP2 vector (42) in front of the luciferase
reporter gene. Mutants in Pit-1 or in AP1 consensus sites of the
sequence P1 were built using the 164P-Luc plasmid and adequate mutated
oligonucleotides (Pit-1 m and AP1 m, see below), with the Chameleon
double-stranded, site-directed mutagenesis kit from Stratagene (La
Jolla, CA). The correct sequence of all these mutant plasmids was
confirmed by dideoxy chain termination sequencing. All plasmids were
prepared by alkaline lysis and purified by centrifugation in
CsCl-ethidium bromide gradients.
Oligonucleotides
Single- and double-stranded oligonucleotides used for plasmid
mutations and gel shift assays were obtained from Eurogentec (Seraing,
Belgium). The AP1 oligonucleotide contains the AP1 site of human
collagenase promoter (43). The CRE oligonucleotide contains the CREB
binding site of the human CG gonadotropin -subunit promoter (44).
The NFY oligonucleotide contains the NFY consensus site of the rat
albumin promoter (45). For each oligonucleotide, the sequence of one
strand is presented below:
[Pit-1 m: 5'-CCTTTGATATCTTCACAGATATAATGAATCAGGC-3'
AP1 m: 5'-CTTCATGAATATAATGCAGCAGGCATTCGTTTCCC-3'
P1: 5'-CTAGAATGCCTGAATCATTATATTCATGAAGATATC-3'
P1, Pit-1 m: 5'-CTAGAATGCCTGAATCATTATATCTGTGAAGATATC-3'
P1, AP1 m: 5'-CTAGAATGCCTGCTGCATTATATTCATGAAGATATC-3'
P2: 5'-CTTCCTGAATATGAATAAGAAATAAAATACC-3'
AP1: 5'-AGCTTAAAGCATGAGTCAGACACCT-3'
CRE: 5'-CTAGAAATTGACGTCATGGTAA-3'
Oct-1: 5'-TGTCGAATGCAAATCACTAGAA-3'
NFY: 5'-GGGGTAGGAACCAATGAAATGAAAGGTTA-3'
Cell Cultures and Electroporation
GH3B6 cells were grown in monolayer, in DMEM (GIBCO-BRL, Grand
Island, NY) supplemented with 10% FCS (GIBCO-BRL), 1%
penicillin/streptomycin solution (GIBCO-BRL), and 5 x
10-10 M estradiol (E2, Sigma). For
transient transfection experiments, cells were harvested using
trypsin-EDTA and resuspended in DMEM supplemented with FCS and
E2, at a concentration of 15 x 106
cells/ml. Then, 800 µl of cell suspension were mixed with 6 pmol of
plasmid and submitted to a single pulse of 250 V/4 mm and 1500
µFarads capacitance, using a "cellject" apparatus (Equibio,
Seraing, Belgium). Electroporated cells were transferred in six-well
tissue culture dishes and treated 3 h later with the appropriate
drugs for about 18 h. Cells were then lysed in 25 mM
Tris/phosphate, pH 7.8; 8 mM MgCl2; 1
mM EDTA; 1% Triton X-100; 15% glycerol; 1 mM
dithiothreitol (DTT). Protein concentrations were determined using the
Bradford assay (46). Before lysis of the cells, toxicity of treatments
was tested using the trypan blue exclusion assay.
Cell Extracts and Western Blot Assay
Nuclear cell extracts were prepared from OA-treated or control
cells. The collected cells were incubated for 15 min in hypotonic
buffer ( 10 mM HEPES; 10 mM KCl; 0.1
mM EDTA; 0.1 mMEGTA; 1 mM DTT; 0.5
mM phenylmethylsulfonylfluoride) before addition of 25 µl
of NP40 10%. After centrifugation for 30 s at 14,000 rpm, the
pellet was resuspended in binding buffer (20 mM HEPES; 400
mM KCl; 20% glycerol 20%; 2 mM DTT)
containing an antiprotease mixture (Boehringer Mannheim, Indianapolis,
IN). Then it was frozen in liquid nitrogen, thawed, and submitted to a
second centrifugation for 30 min at 14,000 rpm. The supernatant was
stored at -70 C, and the protein concentration was determined using a
Bradford assay (46) with BSA as a standard.
Proteins were separated by electrophoresis on a 8% SDS-polyacrylamide gel. After migration, the gel was electroblotted on to a nylon membrane and probed with the appropriate antibody, and the bands were revealed by chemiluminescence (ECL, RPN2106 kit, Amersham, Arlington Heights, IL).
Luciferase Assay
For each sample, protein concentration was normalized in 350
µl of lysis buffer. Luciferase activity was measured in a Lumat LB951
apparatus (EG&G Berthold, Wildbad, Germany) after addition of 100 µl
of a 0.3 mM luciferin/0.8 mM ATP solution.
Results are expressed as the ratio of luciferase activity measured in treated vs. untreated cells. As both treated and untreated cells originate from the same pool of electroporated cells, then variations in luciferase activity are only due to OA treatment. Results are expressed as the mean ± SD of three independent pools of electroporated cells.
Gel Retardation Assays
Gel retardation analysis was performed using 5 µg of nuclear
protein extract, 2.5 µg of poly(deoxyinosinic-deoxycytidylic)acid
and 10,00015,000 cpm of 32P-labeled DNA fragments, in the
following buffer: 5 mM HEPES; 100 mM KCl; 5%
glycerol. The resulting DNA-protein complexes were resolved by
electrophoresis on a prerun 5% native-polyacrylamide gel, with
0.5xTris-borate-EDTA as running buffer. The gel was dried and then
exposed overnight to x-ray sensitive films (Fuji, Stamford, CT).
Nuclear cell extracts were prepared in the same way as the extracts
used in Western blot assays, and double-stranded oligonucleotides were
labeled by the T4 polynucleotide kinase (Pharmacia, Piscataway, NJ)
with [-32P]ATP.
When antibodies were used to reveal the DNA-protein complexes, gel retardation assays were performed as described, except that nuclear extracts were incubated with unlabeled DNA fragments. After migration, gel was electroblotted on to a nylon membrane, probed with anti-Pit-1, anti-junD, or anti-c-fos antibody and revealed by chemiluminescence (ECL, RPN2106 kit, Amersham).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Grant 9381128/A000/DRET/DS/SR from the Direction des Recherches et Etudes Techniques (DRET); Grant PAI P430 from the Services Federaux des Affaires Scientifiques, Techniques et Culturelles; Grants V6/5-ILF.379, -3.4537.93, and -9.4569.95 from the Fonds National de la Recherche Scientifique (FNRS); Grant 95/00193 from Actions de Recherche Concertees; and Grant ASTF 8413 from the European Molecular Biology Organization (EMBO).
1 Equal contributors to this manuscript.
Received for publication February 4, 1998. Revision received April 27, 1998. Accepted for publication April 28, 1998.
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REFERENCES |
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