From the
The effects of a phorbol ester (TPA) and of members of the Jun
and Fos oncoprotein family on the activity of the rat
The
The AFP gene is mainly regulated at the
transcriptional level. It is now clear that the promoter region confers
the liver specificity of expression and that upstream positive and
negative regulatory elements, which are strongly liver-specific, can
also control the transcription rate of the AFP gene (see Refs.
2-4 for reviews). Several of the liver-enriched transcription
factors which may participate in the functioning of the AFP promoter
and enhancers in the liver cell have been characterized
(5, 6, 7, 8, 9, 10, 11, 12) .
Transfection experiments showed that the activities of the rat AFP
promoter and enhancer at
The proteins of the
Jun family are transcription factors involved in several basic cellular
activities which govern differentiation and proliferation and in
several signal transduction pathways (see Refs. 21-24 for
reviews). These proteins all belong to the class of the basic leucine
zipper proteins that bind DNA as dimers. These dimers may be
homodimers, but are generally heterodimers formed between two members
of the Jun family or mainly in association with members of the Fos
family that have a stronger affinity for DNA.
Formation of Jun-Fos
(AP1) complexes is the last step in a cascade of events which allows
transmission of several types of signals from the membrane to the DNA
transcriptional regulatory elements of target genes. For instance, AP1
complex mediates specific transcriptional effects following the
activation of membrane receptors (Ras) and that of protein kinases
(protein kinase C) in response to growth factors or chemicals such as
phorbol esters (TPA). Several activation steps which involve
phosphatases and kinases lead to functional Jun-Fos complexes that act
upon binding to DNA sequences whose consensus is TGAC/GTCA (see Refs.
21-24 for reviews).
Jun proteins can also participate in the
cross-talk between independent regulatory pathways. For instance,
Jun-Fos complexes have been shown to interfere with the cAMP signal
transduction pathway because the binding sites for members of the CREB
family (consensus TGACGTCA) are very similar to those for the AP1
complex. Jun may also be involved in the negative regulation induced by
glucocorticoids. More generally, regulatory interactions of Jun with
several steroid-thyroid hormone receptors can occur through mechanisms
which are still debated (see Refs. 25-27 for reviews). Jun may
also interact directly with some tissue-specific transcription factors
of the MyoD
(28, 29) and C/EBP families
(30) .
Very recently, c-Jun was found to participate in the modulation of
certain liver-specific regulatory elements, such as the
phosphoenolpyruvate carboxykinase promoter
(31) , the albumin
far-upstream enhancer
(32) , and the hepatocyte nuclear factor 1
(HNF1) promoter
(33) . It may also participate in the general
mechanisms which allow correct development of the liver
(34) or
modulation of liver gene expression during liver regeneration
(35) .
The present study was undertaken to determine whether
Jun participates directly or indirectly in the modulation of some of
the rat AFP regulatory elements in the context of an hepatoma cell line
which expresses the AFP gene. Transient transfection experiments were
performed to monitor the effects of TPA and overexpressed Jun on the
activity of vectors bearing the chloramphenicol acetyltransferase (CAT)
gene under the control of the rat AFP promoter region and of its 7 kb
of 5` end-flanking sequences.
The results show that TPA has a strong
negative effect on the AFP promoter in the context of the HepG2
hepatoma cells. c-Jun plays a major role in this highly
promoter-specific inhibition. Interestingly, c-Jun appears to act
indirectly via a mechanism which does not require its binding to a
precise DNA region of the AFP promoter and which involves the
N-terminal part of the c-Jun protein.
Plasmids
Plasmid
pALB-CAT was obtained by cloning the
CAT plasmids bearing the thymidine kinase (TK)
promoter of the herpes simplex virus and one copy of a functional or a
mutated TPA-responsive element (TRE) of the collagenase gene,
pTRE-TK-CAT and p
The plasmids pF1-, pF2-, pF3-, and pF4-SV-LUC were
constructed in a similar fashion, except that the plasmid pGL2-promoter
(Promega) containing the SV40 promoter in front of the luciferase gene
was used instead of plasmid pTK-LUC.
The control plasmids
pTRE-TK-LUC and p
The resulting plasmids were checked by sequencing using
the dideoxy method.
Co-transfection experiments were
performed with increasing amounts (0-5 µg) of the expression
vectors while keeping the total amount of transfected DNA at 12 µg
by adding pUC18 DNA. The precipitate was left in contact with the cells
for 4 h. Cells were then submitted to a 20% glycerol shock for 2 min,
and the medium was changed. When desired, TPA dissolved in 10 µl of
1% Me
The CAT reaction was routinely monitored on samples containing
50-100 µg of protein from HepG2. CAT activity was measured
either after separation of [
The plasmid pCH110, coding for
HepG2 cells were transfected with the luciferase plasmids
in the same way, except that 5 µg of plasmids bearing the
luciferase gene were used, together with 5 µg of the pRSV c-Jun
expression vector or with 5 µg of pUC18 plasmid. Cells were lysed
by adding 400 µl of the Reporter lysis buffer (Promega), incubation
for 15 min at room temperature, and scraping. Luciferase activity was
recorded for 30 s in a Berthold Lumat LB 9501 luminometer for samples
containing 20 µg of protein, using the protocol and reagents given
in the Luciferase Assay System (Promega).
All the transfection
experiments were repeated 3-8 times, usually in duplicate, with
at least two different preparations of the purified plasmids. DNase 1 Footprinting Experiments DNA probes for DNase 1 footprinting assays were end-labeled by filling
in cohesive ends using [
Purified recombinant
human c-Jun protein was purchased from Promega. DNase 1 footprinting
assays were performed as described
(8) , except that the binding
reaction was done in the buffer suggested by Promega, with 1 µg of
poly(dI-dC)-poly(dI-dC) as a nonspecific competitor DNA. The
specificity of the protections was verified by competition experiments
using oligonucleotides carrying an AP1 consensus binding site or
unrelated sequences.
Thus, TPA
specifically decreases the activity of the AFP promoter, in HepG2
cells, but not that of the albumin promoter.
The promoter specificity and
magnitude of the response observed with c-Jun were the same as that
induced by TPA. This strongly suggested that the effect of TPA is
mainly mediated by c-Jun under our conditions. These results clearly
demonstrate that c-Jun can participate in the modulation of the AFP
promoter activity in HepG2 hepatoma cells.
We observed that TPA
treatment of the cells can further enhance the down-regulating effect
of the overexpressed Jun (data not shown). This strongly suggests that
the TPA-mediated stimulation of the cellular protein kinase C induces
modifications (phosphorylation) of the overexpressed c-Jun which result
in the reinforcemenet of its negative action on the AFP promoter. Thus,
although the overexpressed c-Jun may not be present in its fully active
form in the absence of protein kinase C stimulation, it acts in the
same way as a cellular Jun molecule that has been
``activated'' by TPA.
We used a similar approach to show
that two other members of the Jun family of transcription factors, JunB
and JunD, also acted on the AFP promoter and decreased its activity
(Fig. 3). However, assuming that the same quantities of
functional proteins were produced by the three expression vectors, JunB
and JunD were poorer inhibitors of the AFP promoter in HepG2 cells than
was c-Jun.
We used another approach to locate the region(s) where
c-Jun exerted its action. Overlapping fragments of the AFP promoter
known to bind regulatory proteins were cloned into luciferase plasmids
containing the TK promoter instead of the AFP promoter. Each of these
fragments,
The F1 to
F4 fragments of the rat AFP promoter were also cloned into luciferase
plasmids containing the SV40 promoter instead of the TK promoter and
used in transient expression experiments in HepG2 cells. None of these
F1 to F4 fragments inhibited the stimulatory effect that c-Jun exerted
on the basic pSV-LUC plasmid upon binding to the AP1 site present in
the SV40 promoter (data not shown).
The present study demonstrates that TPA and members of the
Jun family of transcription factors can specifically block the activity
of the rat AFP promoter in HepG2 hepatoma cells. These effects are
specific to the AFP promoter, as neither the liver-specific albumin
promoter nor the ubiquitously active TK promoter were modulated by TPA
or c-Jun when assayed under the same conditions. This specificity
excludes the trivial risks of ``squelching'' or the use of a
cryptic Jun responsive element in the vector
(45, 46) .
c-Jun was found to be more potent than JunB or JunD in
down-regulating the AFP promoter. Other examples of such a hierarchy in
the members of the c-Jun family have been reported for several
biological actions
(47, 48, 49) . c-Fos never
acted in synergy with c-Jun to block the AFP promoter activity.
The
specificity of the negative response of the rat AFP promoter to members
of the Jun family is reminiscent of an elegant study which showed that
Ras can down-regulate the human AFP promoter in HuH7 hepatoma cells,
but not the albumin promoter
(17) . As in our case, the exact
region of the human AFP promoter involved in this response to Ras could
not be precisely mapped. The results suggested that the suppressive
effect of Ras is mediated by one or more elements within the 169-bp
region upstream of the transcription initiation site. We now know that
one of the effects of Ras is to stimulate the c-Jun activity in several
cell types. It may well be that the effects of Ras on the AFP promoter
involve c-Jun. Supporting this hypothesis, the same group has very
recently reported that c-Jun can down-regulate the AFP promoter in HuH7
cells
(50) . These results therefore support the notion that a G
protein-linked signaling cascade, or other pathways acting through the
Jun family of transcription factors, might be involved in the
differential regulation of the AFP and albumin promoters during normal
development and in liver cancer. We have, similarly, shown that the AFP
promoter can use several strategies, including the use of HNF1
The mechanisms leading to negative
regulation in eukaryotes may be of several types and are very often
complex (see Ref. 51 for review). Those involving c-Jun are well
documented (see Refs. 25-27 for reviews). Here, it appears that
the down-regulation of the AFP promoter by c-Jun does not involve the
binding of c-Jun to a precise region of the AFP promoter. Our DNase 1
footprinting experiments showed no high affinity binding site for c-Jun
on the AFP promoter. The transfection experiments showed that c-Jun
proteins, mutated in their DNA binding domains, retained the ability to
down-regulate the AFP promoter. That c-Fos did not enhance the c-Jun
effect is in agreement with these findings. Indeed, if binding of Jun
to DNA were involved, one might have expected a synergy between c-Fos
and c-Jun in repressing the AFP promoter because the affinity of the
Jun-Fos heterodimer for DNA is stronger than that of the Jun homodimer.
Lastly, the experiments with the Jun-Gal4 chimera clearly showed that
the N-terminal part of the c-Jun protein which contains the activating
domain is sufficient for the negative effect.
We cannot definitively
rule out the possibility that TPA or c-Jun stimulate the
expression/activity of an inhibitory factor or repress that of a
trans-activator. However, we believe that the effect of Jun on
the AFP promoter is mainly mediated by negative regulatory interactions
between Jun and another transcription factor. Such interactions may
involve interference with binding to DNA or interference with
transcriptional activation. Since Jun does not need to be bound to DNA
for repression of the AFP promoter activity, it is unlikely that the
mechanism entails interference with binding to DNA. This
down-regulation could require protein-protein interactions between Jun
and another(other) transcription factor(s) which favor the functioning
of the AFP promoter. The fact that c-Fos could not potentiate the
effect of c-Jun, but even seemed to antagonize c-Jun action at some
c-Jun:c-Fos ratios, favors this hypothesis. Fos overexpression would
result in the trapping of c-Jun, which would thus be no longer
available to interact with a positive trans-acting factor on
the AFP promoter and counteract its stimulatory effect.
Other
examples of negative regulation by Jun which do not require the binding
of Jun to DNA, but are mediated through interaction of the N-terminal
activating domain of Jun with MyoD or cardiac co-activators have been
reported
(28, 29) .
Jun is known to also counteract
the action of several steroid/thyroid hormone receptors: the
glucocorticoid (GR), progesterone, and estrogen receptors, the thyroid
hormone receptor, and the retinoid receptors RAR/RXR (see Refs.
25-27 for reviews). GR and RXR, therefore, might have been among
the best candidates for negative interaction with c-Jun on the AFP
promoter. It has been shown that the GR and that the RXR can bind to
regions at
The trans-activator target
for the Jun repression of the AFP promoter was not identified because
our different approaches using the luciferase gene under control of
regions of the rat AFP promoter failed to point out a single short DNA
region of the AFP promoter where Jun exerts its regulatory effect. The
negative effect of c-Jun we observed on the 330-bp fragment of the AFP
promoter may also reflect the sum of several negative effects which
take place at different positions on the AFP promoter. There may be a
negative interaction of Jun with one of the components of the general
transcriptional machinery as just shown for Fos
(54) . A strong
specificity, conferred by the context of the proximal AFP promoter
region should be required however. A parallel can be drawn with studies
showing that another DNA-binding protein, p53 protein implicated in the
control of cell proliferation and tumor progression, can specifically
down-regulate various promoters
(55) . The identification of
cis targets of this negative regulation by p53 has also proved
to be difficult. In fact, minimal promoters containing little more than
a TATA box can be down-regulated. Direct interaction between p53 and
factors of the general machinery of transcription may be responsible
for this negative effect (see Ref. 51 for review). Here again, the
exact mechanism is not known and the question of how the promoter
specificity of the response is achieved remains open.
Answers to
these questions are of crucial importance for a better understanding of
how powerful and versatile proteins such as the Jun proteins are
involved in cross-talk between tightly regulated pathways so as to
achieve specific goals within a given cell and promoter context.
We sincerely thank Drs. B. Binetruy, D. Bohmann, and
M. Yaniv for their gifts of essential plasmids. We are grateful to Dr.
M. Yaniv for his interest in the work. We also thank Dr. O. Parkes for
his help in correcting our English.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-fetoprotein
(AFP) promoter were checked by using transient expression experiments
in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP
promoter in a dose-dependent manner. Overexpression of c-Jun
specifically repressed the rat AFP promoter but not the albumin
promoter. JunB and JunD were poorer inhibitors. c-Fos expression did
not potentiate the negative effect of Jun. The Jun-induced repression
does not require binding of c-Jun to the AFP promoter. DNase 1
footprinting experiments did not display any high affinity binding site
for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain
is not required for the c-Jun protein to block the AFP promoter. The
N-terminal part of Jun, which contains the activating domain, is
responsible for the repression as shown by using Jun-Gal4 chimera. Jun
likely exerts its negative control on the AFP promoter via
protein-protein interactions with a not yet identified
trans-activating factor within the
134 to +6
region or with a component of the general machinery of transcription.
Jun proteins can thus be key intermediates in regulatory cascades which
result in the differential modulation of the AFP and albumin gene
expression in the course of liver development and carcinogenesis.
-fetoprotein gene (AFP),
(
)
which
belongs to the same family as the albumin gene, is expressed in a very
tightly controlled manner in the course of mammalian development. It is
specifically expressed at a high rate in the yolk sac, the fetal liver,
and to a lesser extent in the fetal gut.
-Fetoprotein, whose
function is still not fully understood, is the most abundant plasma
protein during the fetal period. Transcription of the AFP gene drops
abruptly around birth and is virtually totally blocked during normal
adult life. Expression of the AFP gene can, however, be resumed under
certain pathophysiological conditions, such as liver regeneration after
partial hepatectomy or chemical injury and in hepatocarcinogenesis (see
Ref. 1 for review). The AFP gene, whose expression is controlled by
multiple signal transduction pathways, is a powerful model with which
to examine the molecular mechanisms that dictate the liver specificity
of gene expression and which modulate its activity during development
and cancerogenesis.
2.5 kb can be down regulated by
dexamethasone
(13, 14, 15) . We also know that
12- O-tetradecanoylphorbol-13-acetate (TPA) and epidermal
growth factor and an oncogenic, mutated form of ras often
found in primary hepatocarcinomas, can reduce the activities of
regulatory elements present in the 5` end extragenic region of the
human AFP gene
(16, 17) . Transgenic studies indicate
that the activities of the mouse AFP regulatory elements can be
stimulated during liver regeneration
(18) , when expression of
AFP
(19) and of several proto-oncogenes is greatly enhanced
(see Ref. 20 for review). The mechanisms underlying these modulations
of the AFP regulatory elements are still largely unknown. However, it
is quite possible that proto-oncogenes of the Jun family play a key
role in some steps of these cascade mechanisms.
CAT Plasmids
Plasmids pBL-CAT2 and -3
(36) were
used as starting material to construct plasmids bearing the CAT gene
under the control of rat AFP or albumin gene promoters. The
SacI/ XbaI 1-kb fragment (named UMS) from the upstream
region of the mouse c- mos gene was cloned just in front of the
polylinker region of these plasmids (plasmids pBL-UMS-CAT2 and -3) to
prevent the contribution of any spurious initiation of transcription in
the plasmid
(37) . Conventional cloning procedures were used to
insert the 197 to +6 ( BbvI- HpaII)
fragment or the
324 to
15
( HindIII- HaeII) fragment of the rat AFP promoter
(38) into the HindIII- BglII sites of
pBL-UMS-CAT3, yielding plasmids pAFP26-CAT and pAFP-CAT, respectively.
The fragment
7200 to
324
( EcoRI- HindIII) of the 5` end extragenic region of
the rat AFP gene was cloned in the polylinker in front of the AFP
promoter in plasmid pAFP-CAT to give plasmid pPO123-AFP-CAT. Plasmid
pBL-AFP36-CAT was made by cloning the blunted
324 to +6
( HindIII- HpaII) fragment of the rat AFP promoter into
the blunted BglII site of plasmid pBL-CAT6.
175 to +15
( AluI- HincII) region of the rat albumin promoter
(39) at the HindIII- BglII sites in plasmid
pBL-UMS-CAT3.
TRE-TK-CAT, respectively, have been described
previously
(40) .
pAFP-LUC Series
The parent plasmid pAFP-LUC,
containing the luciferase (LUC) reporter gene under the control of the
AFP promoter, was constructed by inserting the blunted
HindIII- HpaII (324/+8) fragment of the
AFP promoter into the blunted BglII site of pGL2-basic
(Promega). The plasmids pAFP-LUC
234,
183,
155,
134,
115,
67, and
49 were generated by
progressively deleting 5` portions of the AFP promoter in the plasmid
pAFP-LUC with exonuclease III/mung bean nucleases.
pTK-LUC and pSV-LUC Series
To obtain plasmid
pTK-LUC, the BamHI- BglII fragment containing the TK
promoter (105/+51) was excised from pBL-CAT5 and inserted
into the BglII site of pGL2-basic. The pF1 to F4-TK-LUC series
was constructed by inserting different fragments of the AFP promoter
upstream of the TK promoter in pTK-LUC. The
BbvI- MaeIII (
196/
135),
MaeIII- MaeIII (
142/
79), and
BbvI- HincII (
196/
52) fragments of the
AFP promoter were isolated, blunted, and introduced into the blunted
NheI site of pTK-LUC, generating the pF1, pF2, and pF4-TK-LUC
plasmids, respectively. A blunt-ended oligonucleotide encompassing the
85- to
41-bp region of the AFP promoter was cloned into
the blunted NheI site of pTK-LUC to generate plasmid
pF3-TK-LUC.
TRE-TK-LUC were generated by transferring a
fragment carrying the wild type or mutated TPA-responsive element of
the collagenase gene and the TK promoter from the pTRE-TK-CAT or
p
TRE-TK-CAT plasmid, respectively, to pGL2-basic. The TRE-TK or
TRE-TK fragment was excised with HindIII- BglII,
blunted, and inserted into the blunted BglII site of
pGL2-basic.
Jun and Fos Expression Vectors
Vectors allowing
the expression of mouse c-Fos (pRSV c-Fos), mouse c-Jun (pRSV c-Jun), a
mouse c-Jun lacking its N-terminal activating domain (pRSV c-Jun
168) or its leucine zipper region (pRSVc-Jun CDL), and those for
JunB and JunD (pRSV JunB, pRSV JunD) were kindly donated by M. Yaniv
(41) . Vector allowing the expression of human c-Jun was a gift
from B. Binetruy and M. Karin. Vectors allowing the expression of human
c-Jun proteins with point mutation in the DNA binding domain (pRSV
c-Jun-DB3 and pRSV c-Jun-DB4, mutants 12 and 14 in Ref. 42) and that of
a chimeric protein made with the N-terminal part of c-Jun (amino acids
1-253) fused to the DNA binding domain of Gal4 (pDB10) were gifts
from D. Bohmann
(42) . Transfection and Transient Expression Experiments Human hepatoma cells HepG2 were obtained from the American Type Culture
Collection and grown in a 1:1 mixture of Dulbecco's modified
Eagle's medium and Ham's F12 media containing 10% fetal
calf serum. Transfection experiments were performed in 6-cm plastic
tissue culture dishes containing about 10
exponentially
growing cells using the calcium phosphate method. 2 µg of CAT
plasmid were routinely used.
SO was then added. 48 h later, cells were lysed by
three successive freeze-thaw methods in 100 µl of 0.25 M
Tris-HCl buffer, pH 7.8. The homogenate was centrifuged for 10 min at
11,000
g, and the clear supernatant was frozen at
20 °C. Protein concentrations were determined by the
Bradford method with bovine immunoglobulin as standard (Bio-Rad).
C]chloramphenicol
from the acetyl [
C]chloramphenicol by TLC or
from the butyryl [
C]chloramphenicol by
extraction into tetramethyl- p-phenylenediamine/xylene
(43) . Both methods gave the same results in our hands. Under
our conditions, the activity of pAFP-CAT in the HepG2 cells was usually
about 8-10% of that of plasmid pSV2-CAT, and that of pALB-CAT was
3%. The activity of the promoterless plasmid pBL-UMS-CAT3 was close to
background and was not significantly altered by expression of any of
the transcription factors used.
-galactosidase (2 µg), was used in some control experiments to
monitor the efficiency of transfection. This plasmid was not routinely
included in the co-transfection experiments because its activity was
greatly affected by TPA and by expression of some of the transcription
factors.
-
P]dATP and Klenow
polymerase. Probes were purified by polyacrylamide gel electrophoresis
followed by electroelution. A 340-bp DNA probe encompassing the AFP
promoter was isolated from a plasmid pBluescript SK+ (Stratagene)
carrying the blunted HindIII- HpaII fragment of the
AFP promoter (
324/+8) inserted into the EcoRV
site. The AFP promoter was labeled on either the antisense or the sense
strand using the EcoRI or the HindIII site situated
in the pBluescript polylinker. A 227-bp DNA fragment containing the
TPA-responsive element of the collagenase promoter and part of the TK
promoter was labeled on the sense strand at the EcoRI site
internal to the TK promoter and subsequently isolated from the
pTRE-TK-CAT plasmid by NarI digestion.
Inhibition of the AFP Promoter in HepG2 Cells by
TPA
The action of TPA on the activities of AFP regulatory
elements in the promoter of the rat AFP gene and in its 7-kb 5` end
extragenic region was examined by monitoring the CAT activity of
plasmids pPO123-AFP-CAT and pAFP-CAT transiently expressed in HepG2
cells. The activities of both plasmids were greatly reduced
(75-88%) when HepG2 cells were treated with TPA
(Fig. 1 A). The basal activity of the pPO123-AFP-CAT
plasmid was much more higher (about 8-fold) than that of pAFP-CAT, in
agreement with the fact it contains several enhancer elements acting on
the AFP promoter
(14) . The effect of TPA on the activity of
several other CAT plasmids was also assessed under the same conditions,
as controls. The promoterless plasmid pBL-UMS-CAT had very little CAT
activity (near background) that was not altered by TPA. The activity of
the plasmid pTRE-TK-CAT, which contains one copy of the TPA-responsive
element (TRE) of the collagenase gene in front of the TK promoter, was
stimulated by TPA, as expected. In contrast, the activity of
pTRE-TK-CAT, which contains the TRE element with a 2-bp deletion
that greatly reduces the binding of Jun, was not affected
(Fig. 1 A).
Figure 1:
Negative modulation
of the rat AFP gene promoter by TPA in HepG2 hepatoma cells.
A, HepG2 cells were transfected with 2 µg of CAT plasmid
bearing the rat AFP promoter and 7 kb of the rat AFP gene 5` end
extragenic region ( pPO123-AFP-CAT) or the rat AFP promoter
alone ( p-AFP-CAT), 2 µg of plasmids containing the TK
promoter, and a single copy of the TPA responsive element (TRE) of the
collagenase gene ( pTRE-TK-CAT) or a mutated TRE
( pTRE-TK-CAT) were used as controls within the same batch
of experiments. CAT activities were monitored 48 h later for HepG2
cells treated with 50 ng of TPA in 10 µl of 1%
Me
SO(+) or 10 µl of 1% Me
SO alone
(
). B, 2 µg of CAT plasmids bearing the AFP
promoter ( pAFP-CAT) or the albumin promoter
( pALB-CAT) were transfected into HepG2 cells treated with
increasing amounts of TPA in 1% Me
SO or with 1%
Me
SO alone. CAT values corresponding to plasmid pAFP-CAT
(
) or pALB-CAT (
) are expressed with reference to those
obtained with the corresponding plasmids in the absence of
TPA.
Although we could not exclude that the
region from 7000 to
330 of the rat AFP gene does not
contain negative TPA responsive elements, we focused on the AFP
promoter. Fig. 1 B shows that the negative effect of TPA
on the AFP promoter activity was dose-dependent. This effect also
appeared to be specific to the AFP promoter, since the activity of
pALB-CAT, which contains the albumin promoter region (
175 to
+15) instead of the AFP promoter in the same pBL-UMS-CAT vector,
was unaffected by TPA over the same range of doses.
Negative Regulation of the AFP Promoter in HepG2 Cells by
Jun Transcription Factors
The effect of Jun on the AFP promoter
activity was checked by co-transfecting HepG2 cells with the pAFP-CAT
plasmid plus increasing amounts of plasmid pRSV c-Jun, which allows
expression of the mouse c- jun proto-oncogene. The activity of
plasmid pAFP-CAT was lowered in a manner dependent on the amount of the
c-Jun expression vector (Fig. 2). It was inhibited to
12-15% of its basal value. Similar results were obtained with a
pRSV vector allowing expression of the human c-Jun protein (data not
shown).
Figure 2:
c-Jun
down-regulates the rat AFP promoter activity in HepG2 hepatoma cells.
HepG2 cells were transfected with 2 µg of CAT plasmid bearing the
rat AFP promoter (pAFP-CAT), the rat albumin promoter (pALB-CAT), or
the thymidine kinase promoter of the herpes simplex virus (TK) and the
TRE from the collagenase gene (pTRE-TK-CAT), together with increasing
amounts of a mouse c-Jun expression vector (pRSV c-Jun). The total
amount of transfected DNA was kept constant at 12 µg by addition of
pUC18. CAT activities were measured 48 h after transfection. The
upper part of the figure shows the variation of the activity
of the pAFP-CAT plasmid as a function of increasing amounts of the pRSV
c-Jun plasmid. In the lower part of the figure, activities of
the pAFP-CAT plasmid (), of the pALB-CAT plasmid (
), and of
the pTRE-TK-CAT plasmid (
) are expressed with reference to
those of the corresponding plasmids in the absence of c-Jun expression
vector.
The strong negative effect of c-Jun on the AFP promoter was
highly specific. The expression of c-Jun did not significantly alter
the CAT activity of the plasmid bearing the albumin promoter and had,
as expected, a positive effect on the pTRE-TK-CAT plasmid carrying the
TRE of the collagenase gene. The specificity of the response of these
plasmids also indicated that the negative effect of c-Jun on the AFP
promoter-containing vectors did not result from general blocking of
RNA-PolII-dependent transcription machinery or from unrelated AP1 sites
present in the plasmid
(46) .
Figure 3:
Effects of mouse JunB and mouse JunD on
the rat AFP promoter activity in HepG2 hepatoma cells. HepG2 cells were
transfected with 2 µg of the CAT plasmid bearing the rat AFP
promoter region (pAFP-CAT) together with increasing amounts of plasmids
allowing expression of mouse c-Jun (), JunB (
), or JunD
(
). The total amount of transfected DNA was kept constant at 12
µg by addition of pUC18. CAT activities were measured 48 h later
and are expressed with reference to the activity of the pAFP-CAT
plasmid in the absence of Jun expression
vectors.
Since Jun usually binds to DNA as a heterodimer with Fos,
we tested the effect of the c- fos proto-oncogene alone and in
combination with different amounts of c-Jun on the AFP promoter
activity. HepG2 cells were co-transfected with the pAFP-CAT plasmid and
increasing amounts of c-Fos expression vector. The influence of
different c-Fos:c-Jun ratios was examined by using the c-Fos expression
vector alone or with two quantities of the c-Jun expression vector
(Fig. 4). c-Fos expression alone has very little, if any,
negative effect on the AFP promoter activity. More importantly, c-Fos
never potentiated the negative effect of c-Jun on the AFP promoter. Fos
even seemed to counteract c-Jun at some Fos:Jun ratios. This suggests
that c-Jun, and not a c-Jun-c-Fos complex, is mainly responsible for
down-regulating the AFP promoter in HepG2 cells.
Figure 4:
Effects of c-Fos or combined c-Fos-c-Jun
expression on the rat AFP promoter activity in HepG2 hepatoma cells.
HepG2 cells were transfected with 2 µg of the CAT plasmid bearing
the rat AFP promoter (pAFP-CAT) together with increasing amounts of a
mouse c-Fos expression vector (pRSV c-Fos) alone () or in the
presence of 0,8 µg (
) or 2,5 µg (
) of a mouse c-Jun
expression vector (pRSV c-Jun). The total amount of transfected DNA was
kept constant at 12 µg by addition of pUC18. CAT values were
measured 48 h later and are expressed with reference to that of the
pAFP CAT plasmid in the absence of any expression
vector.
The Repression of AFP Promoter Activity Does Not Require
Binding of c-Jun to DNA and Involves the N-terminal Part of
c-Jun
We determined whether c-Jun can bind to the AFP promoter
by DNase 1 footprinting experiments with purified recombinant human
c-Jun (Fig. 5). A probe containing the TRE-TK promoter was used
as a control. Purified c-Jun did not specifically bind to either of the
two strands of the rat AFP promoter under the conditions where a clear
footprint was observed in the TRE region of the TRE-TK promoter
(Fig. 5). This indicated that c-Jun cannot bind to the rat AFP
promoter with high affinity.
Figure 5:
Purified human c-Jun does not bind with
high affinity to the rat AFP promoter. 1 ng of double-stranded DNA
fragments corresponding to the P-labeled noncoding strand
( middle panel) or coding strand ( right panel) of the
rat AFP promoter and to the coding strand ( left panel) of a
construct containing the TRE of the collagenase gene cloned in front of
the TK promoter (from pTRE-TK-CAT) were incubated without (0) or with
increasing amounts of purified recombinant human c-Jun protein (up to 1
footprinting unit) and then subjected to DNase 1 digestion as described
under ``Materials and Methods.'' Aliquots of G + A Maxam
and Gilbert reactions performed on each of the labeled fragments were
run as molecular weight markers on the denaturating acrylamide gel.
Numbering of the AFP fragment is from the transcription start site
(38). The nucleotide sequence of the protected TRE element in the
TRE-TK probe is written in the left margin. The specificity of
the protections was verified by competition experiments using
oligonucleotides carrying an AP1 consensus binding site or unrelated
sequences.
To determine which part of the c-Jun
protein is required for the negative regulation of the AFP promoter, we
used vectors allowing the expression of mutated c-Jun proteins. The
activity of two mutants of c-Jun (JunDBand
JunDB
) with point mutations in the DNA binding domain which
greatly lowered their affinity for a TRE binding site
(42) , was
monitored. We also tested mutants c-Jun
168 and c-Jun CDL
(41) from which the activating domain or the Leucine Zipper
region had been deleted, respectively (Fig. 6). Mutants lacking
the activating domain or the leucine zipper domain had lost their
capacity to repress the AFP promoter activity. In contrast, the two
c-Jun proteins mutated in their DNA binding domain (DB3 and DB4) were
still able to down regulate the AFP promoter (Fig. 6). Hence, the
integrity of the DNA binding domain of Jun is not required for the
c-Jun protein to exert its negative effect on the AFP promoter in HepG2
cells.
Figure 6:
Effects of mutated c-Jun on the rat AFP
promoter activity in HepG2 hepatoma cells. HepG2 cells were transfected
with 2 µg of the pAFP26-CAT plasmid bearing the AFP promoter, alone
(0) or together with increasing amounts of vectors allowing expression
of a wild type mouse c-Jun protein (pRSV c-Jun) or of a c-Jun protein
deleted from the activating domain (pRSV CDL c-Jun) or from the leucine
zipper (pRSV 168 c-Jun) or bearing point mutations in the DNA
binding domain (pRSV DB3 c-Jun and pRSV DB4 c-Jun). Total amount of
transfected DNA was kept constant to 12 µg by addition of pUC18
DNA. CAT values were measured 48 h later and are expressed with
reference to the activity of the pAFP26-CAT plasmid in the absence of
any expression vector. Results are given as the mean of 4 independent
experiments. Bars represent the mean ± standard
error.
The absence of a high affinity binding site for c-Jun on the
AFP promoter, plus the fact that the integrity of the c-Jun DNA binding
domain is not required for the negative effect, strongly suggests that
the repressive action of Jun on the AFP promoter does not involve a
direct binding of Jun to the AFP promoter. This hypothesis led us to
test the effect of a chimeric protein made of the N-terminal part of
c-Jun, (amino acids 1-253) which contains the Jun activating
domain, fused to the DNA binding domain of the Gal4 transactivator
(42) . The chimeric Jun-Gal4 protein, expressed from plasmid
pDB10, strongly repressed the AFP promoter (Fig. 7). By
opposition, plasmid pDB10, which was obtained from pDB10 by
deleting only the DNA sequences coding for the N-terminal part of Jun,
had no effect on the AFP promoter (Fig. 7). These results clearly
indicated that the N-terminal part of Jun, which contains the
activating domain, is responsible for the down-regulation of the AFP
promoter. Similar results were obtained with another chimeric protein
containing the N-terminal part of c-Jun (amino acids 1-193) fused
to the DNA binding domain of the growth hormone transcription factor 1
(data not shown).
Figure 7:
The
activating domain of c-Jun is sufficient for down-regulating the rat
AFP promoter in HepG2 hepatoma cells. HepG2 cells were transfected with
5 µg of the pAFP26-CAT plasmid bearing the AFP promoter, alone (0)
or together with 1 µg of a vector allowing expression of a wild
type mouse c-Jun protein (pRSV c-Jun) or of a chimeric protein made of
the N-terminal activating domain of c-Jun linked to the DNA binding
domain of the Gal4 protein (pDB10). pDB10 is the same plasmid as
pDB10 except that the DNA sequences corresponding to the N-terminal
part of c-Jun have been deleted. Total amount of transfected DNA was
kept constant at 10 µg by addition of pUC18 DNA. CAT values were
measured 48 h later and are expressed with reference to the activity of
the pAFP26-CAT plasmid in the absence of any expression vector. Results
are given as the mean of 4 independent experiments. Bars represent the mean ± standard
error.
This finding prompted us to use a functional assay
to locate the region of the AFP promoter which is the target for the
indirect c-Jun action. We constructed a series of vectors containing
the luciferase gene under the control of the AFP promoter that had been
progressively deleted from its 5` end by exonuclease III. They were
introduced into the HepG2 cells alone or together with the c-Jun
expression vector. Deleting the AFP promoter from 336 to
134 resulted in a gradual decrease in its activity
(Fig. 8 A). The background level was reached with the
deletion at
115. This decrease in the activity reflects the
progressive loss of binding sites, at least for C/EBP at
280
(8) , for the fetoprotein transcription factor (FTF)
(11) and the glucocorticoid receptor (GR) in the
160
region
(13) , and those for the retinoic acid X receptor (RXR)
and COUP at
135
(44) . This deletion analysis also
confirmed that the
120 region, to which HNF1, nuclear factor 1
(NF1), and the C/EBPs can bind, is crucial for the AFP promoter
functioning
(5, 7, 8, 12) .
Figure 8:
Effects of c-Jun on the activity of
deletion mutants and of subfragments of the rat AFP promoter in HepG2
hepatoma cells. The upper part of the figure shows a scheme of
the rat AFP promoter with the position of binding sites for
transcription factors which have been previously mapped using gel-shift
or footprinting experiments. A, HepG2 cells were transfected
with 5 µg of a plasmid bearing the luciferase gene under the
control of the rat AFP promoter region in its whole 330/+8
(pAFP-LUC) or truncated at
234,
183,
155,
134,
115,
67,
49, in the absence (
)
or in the presence (
) of 5 µg of the pRSV c-Jun vector
allowing expression of the mouse c-Jun. The total amount of transfected
DNA was kept constant at 10 µg by addition of pUC18. Luciferase
activity was determined 48 h later as described under ``Materials
and Methods,'' and values are expressed as relative light units
( RLU) integrated for 30 s for samples containing 20 µg of
protein. Results are given as the mean of 6 to 9 independent
experiments. Bars represent the mean ± standard error.
B, one copy of fragment
196/
135 (F1),
142/
79 (F2),
85/
41 (F3), or
196/
52 (F4) of the rat AFP promoter was cloned in front
of the TK promoter in the pGL2-TK-LUC plasmid. The effect of c-Jun was
checked on 5 µg of each of the resulting F-TK plasmids introduced
into HepG2 cells in the absence (
) or presence (
) of
the pRSV c-Jun vector exactly as described
above.
The
luciferase activities of the plasmids carrying the intact 324
AFP promoter and its
234,
183,
155, and
134
deletion derivatives were all greatly lowered by expression of the
c-Jun protein. Consequently, any region between
324 and
134 is clearly not needed for the negative effect. However, they
can always be targets for a c-Jun action. The results also strongly
suggested that one or more elements in the region between
134
and +6 are involved in the repression of the AFP promoter activity
by c-Jun.
196/
52,
196/
135,
142/
79,
85/
41, slightly stimulated the TK
promoter activity in the HepG2 cells (Fig. 8 B). However,
the activity of none of these F1-to-F4 TK plasmids was blocked by
overexpression of c-Jun (Fig. 8 B). This indicated that
none of these fragments, when present in a single copy, is able to
confer the c-Jun negative regulation on the TK promoter.
rather than HNF1
and competition between HNF1 and NF1, to
specifically regulate its activity in a way different from that of the
albumin promoter
(12) .
160 and
135 of the rat AFP promoter,
respectively. Upon addition of their respective ligands, GR
down-regulates
(13, 14, 52, 53) and RXR
stimulates
(44) the activity of the AFP promoter. Our
experimental results, however, do not confer the major role to
interactions between Jun and the GR or the RXR in the down-regulation
of the AFP promoter by Jun. Deletions of the regions bearing their
binding sites on the AFP promoter let the remaining promoter still able
to negatively respond to Jun. This indicates that Jun can act
downstream in the AFP promoter upon interaction with protein(s) other
than the GR or the RXR. It is, however, possible that interactions
between Jun and the GR or RXR might play a significant role in the
molecular mechanisms which govern the response of the AFP promoter to
glucocorticoids or retinoids.
-fetoprotein; TPA,
12- O-tetradecanoylphorbol-13-acetate; C/EBP,
CCAAT/enhancer-binding protein; HNF1, hepatocyte nuclear factor 1; CAT,
chloramphenicol acetyltransferase; TK, thymidine kinase; TRE,
TPA-responsive element; LUC, luciferase; GR, glucocorticoid receptor;
RXR, retinoic acid X receptor; NF1, nuclear factor 1; kb, kilobase(s);
bp, base pair(s).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.