From the
Moloney murine leukemia virus (Mo-MuLV) is a thymotropic and
leukemogenic retrovirus which causes T lymphomas. The long terminal
repeat (LTR) of Mo-MuLV affects the regulation of a number of cellular
genes, including collagenase IV, monocyte chemoattractant protein-1,
and c-jun genes, all of which contain
12-O-tetradecanoylphorbol-13-acetate-responsive ele-ment
consensus sites within their promoters. We report here that Mo-MuLV
stimulates the collagenase IV gene through transcription factor AP-1,
and that the expression of a subgenomic portion of Mo-MuLV LTR alone is
sufficient for this effect. Transient or stable expression of the viral
LTR increases cellular AP-1 DNA binding activity. The collagenase IV
12-O-tetradecanoylphorbol-13-acetate-responsive element
consensus sequence was shown to be required for this
trans-activation. Deletions or mutations of this consensus
site which abolished AP-1 binding also abolished
trans-activation by the LTR. Transient or stable transfection
of the viral LTR into cells stimulated c-jun gene expression,
suggesting one mechanism whereby the viral LTR may induce cellular AP-1
activity. Thus, the Mo-MuLV LTR, through activation of the
transcription factor AP-1, is capable of regulating cellular gene
expression, including the induction of proto-oncogenes. This activity
may be relevant to the mechanisms whereby retroviruses which do not
contain oncogenes induce neoplasia.
Moloney murine leukemia virus (Mo-MuLV)
The LTR sequences of murine leukemia viruses are not
known to encode trans-acting viral functions, yet have been
implicated in the etiology and progression of retroviral
diseases
(5, 6, 7, 8, 9) . The
MCF-MuLV LTR, for example, is an important contributor to viral
leukemogenicity in AKR mice
(11) . The U3 region of BL/VL3 viral
LTR has the ability to induce cellular suppressor factors, and generate
intracellular immunity against itself and other MuLVs
(9) .
Deletions within the LTR alter disease specificity
(7) , and
analysis of the SL3-3 AKV recombinant provirus showed the
determinant of leukemogenicity to lie within the SL3-3
LTR
(12) .
We report here that infection of Balb or HeLa cells
with Mo-MuLV, or transfection with vectors expressing regions of the
viral LTR, results in induction of transcription factor AP-1 activity,
as assayed on electrophoretic mobility shift assays (EMSA). This
activation of AP-1 results in increases in transcription of a number of
cellular genes, including collagenase IV, monocyte chemoattractant
protein-1 (MCP-1, also called JE), and c-jun, all of which
contain TRE (TPA responsive element) consensus sequences in their
promoters. Subgenomic portions of the murine leukemia virus containing
the LTR are sufficient to produce activation of transcription factor
AP-1. The transient co-transfection of an LTR-expressing vector
together with chimeric genes containing a collagenase promoter with an
AP-1 binding consensus sequence driving the reporter gene CAT results
in increases of CAT activity. Site-specific mutagenesis of this TRE
binding site prevents binding of the LTR-induced complex and induction
of the reporter gene. EMSAs performed in the presence of anti-Jun or
anti-Fos Family antibodies demonstrate that the AP-1 complex induced by
Mo-MuLV consists of Jun/Fos heterodimers.
We have shown that stable infection of Balb/c-3T3 cells with
Mo-MuLV (MoBalb cells) results in the induction of transcripts for
specific cellular genes, including MCP-1 and collagenase IV, and an
increase in their respective protein products (3).
We have previously demonstrated that infection of human or
murine cells with murine leukemia viruses rapidly increases the
transcription and expression of a number of genes belonging to the
immunoglobulin superfamily and involved in T-lymphocyte activation,
including the Class I MHC
antigens
(1, 2, 3, 4) . More recently, we
have reported that the LTR of Moloney murine leukemia virus encodes a
trans-activator which induces transcription and expression of
MHC Class I genes, T cell activation antigens, and certain cytokine
genes. The portion of the LTR responsible for trans-activation
was mapped by deletions to lie within the U3 region
(3) . We have
also demonstrated that a transcript is initiated by RNA polymerase III
within the U3 region, and its presence correlates with the
trans-activating activity.
The transcriptional response to TPA is mediated in many
cases by the AP-1 transcription factor, which recognizes and binds to a
specific DNA sequence motif that functions as cis-acting
promoter element (23). Induction of AP-1 activity by the viral LTR was
demonstrated during transient or stable expression of the LTR. We have
also shown that the ability of a mutated LTR to trans-activate
AP-1-dependent CAT activity correlates both with the ability of that
LTR to generate a transcript, and with the ability to activate cellular
AP-1 DNA binding activity. The mutant LTR contained in the vector
pGUXDP is incapable of generating the polymerase III-directed
transcript previously demonstrated to be necessary for
trans-activation of a number of cellular genes.
The
transcriptional activation of endogenous cellular genes by the LTR of
Mo-MuLV represents a novel way in which retroviruses can control the
expression of genes in the cells they infect. The mechanisms through
which this activation occurs are beginning to be elucidated. Our
finding that a number of TPA-inducible genes, including MCP-1,
collagenase IV, and c-jun,are induced in response to
the viral LTR led us to determine which cis-acting elements of
these gene promoters are required for induction by the viral LTR. The
promoter region of MCP-1 harbors the sequence TGAGTCC, which resembles
the consensus site for AP-1 binding (TPA-response element, TRE), and
TGAGTCA, which is also present in the collagenase IV gene promoter. The
c-jun gene promoter also contains the sequence TGAGTCA,
raising the possibility that the c-jun gene can be positively
autoregulated by AP-1 through its TRE site
(19) .
Trans-activation of the collagenase IV gene by TPA and by the
LTR both required a TRE site in the promoter. The Mo-MuLV LTR
trans-activated a collagenase IV promoter-CAT construction
which contained an intact TRE site, but the level of
trans-activation by the LTR was significantly lower for the
same promoter-CAT vector with either a 2-base pair deletion or 2-base
pair transversion of the AP-1 binding site. These same mutations were
shown in EMSAs to be unable to bind TPA- or LTR-induced AP-1 complexes.
Therefore, an intact TRE site apparently is required for the
trans-activation of certain genes by the viral LTR. Gel-shift
assays using a TRE consensus oligo supported the hypothesis that the
viral LTR could activate an AP-1 complex which bound to the TRE site.
The DNA binding activity induced by the integrated or transiently
expressed LTR in cells also appeared identical in its binding site
sequence requirements and mobility to the AP-1 complexes induced by TPA
treatment.
The activity of the transcription factor AP-1 is
modulated at least four levels
(24, 25, 26) : 1)
the regulation of the levels or location of c-Fos or c-Jun
(27) ;
2) the regulation of the DNA binding activity of the transcription
factor
(28, 29) ; 3) the modulation of the
trans-activating potential of
c-Jun
(30, 31, 32, 33) , through
post-translational modulation of the protein; and 4) through regulation
of dimerization
(34) .
Expression of the viral LTR elevated
the level of AP-1 DNA binding activity coincidentally with a marked
increase in the expression of c-jun transcripts. In contrast
to c-jun mRNA, increases in c-fos transcripts were
minimal. c-jun is also an AP-1-inducible gene, so its
transcriptional up-regulation may be the result, rather than the cause,
of AP-1 activation by the LTR
(35, 36) . It was therefore
conceivable that the AP-1 activity induced by the LTR might consist
predominantly of Jun/Jun homodimers rather than the Fos family/Jun
family heterodimers induced by TPA. The use of antibodies specific for
the components of AP-1 in EMSA demonstrated, however, that both Jun and
Fos family members were present in the AP-1 complexes induced by the
LTR. It is possible that the basal cellular level of pre-existing c-Fos
proteins is greater than c-Jun proteins, so that the sudden increase in
c-jun driven by the LTR enables the formation of more
c-Jun/c-Fos heterodimers, but this remains to be determined. Changes in
intracellular redox potentials may regulate AP-1
activity
(37, 38, 39) . Reducing agents like
pyrrolidine dithiocarbamate and N-acetylcysteine activate AP-1
activity in HeLa and Balb cells, and the presence of the LTR does not
result in a further increase in AP-1 activity,
The dichotomy between the strong
induction of c-jun transcription and lack of c-fos induction by the viral LTR may provide some information regarding
mechanisms of action of the LTR. Both the c-fos and c-jun genes are induced by exposure to phorbol ester, but the
cis-acting elements controlling this response are distinct.
The AP-1 complex appears to bind directly to c-jun promoter,
but phorbol esters may act indirectly on the c-fos promoter,
through the serum response element. While c-jun transcription
is rapidly induced by many stimuli, its pattern of expression is not
identical to c-fos. For example, ultraviolet (UV) light is a
very potent inducer of c-jun transcription (mediated by a
divergent AP-1 binding site in the c-jun promoter), but UV
light only modestly induces expression of c-fos(40) .
The AP-1 complex, through its cognate DNA-binding site, may serve to
integrate signaling from a number of diverse stimuli, by
transcriptionally activating genes resulting in proliferation,
activation, tumor promotion, transformation, or protection from
apoptosis. Changes in phosphorylation state may regulate AP-1 activity.
The activation of AP-1 by growth factors and tumor promoters is in many
cases through activation of protein kinase C
(41) . The growth
factor receptor- or phorbol ester-stimulated protein kinase C-mediated
pathway may activate AP-1 indirectly by altering the phosphorylation
status of the Jun family of
proteins
(28, 29, 42, 43, 44, 45, 46, 47) ,
allowing dimerization and DNA binding by the complex to occur. More
recently, another protein kinase which is activated by phorbol esters
and phosphorylates c-Jun has been described
(48) . We have shown
that the protein kinase C inhibitor staurosporine and the protein
serine/threonine phosphatase inhibitor okadaic acid exert opposite
effects on Mo-MuLV LTR induction of AP-1 DNA binding activity. These
findings suggest cellular serine/threonine kinase activity may play a
role in the Mo-MuLV LTR stimulation of AP-1 activity.
We and others
have reported the activation by the retroviral LTR of cellular genes
which are not directly dependent on AP-1 activity, and which do not
contain consensus TRE binding sites in their promoters, including MHC
antigens and T cell activation antigens. We have identified by deletion
and footprinting studies another cis-acting element which
appears responsive to activation by the retroviral LTR and which may be
responsible for the trans-activation of some of these genes
(3).
Murine leukemia viruses cause
lymphoid neoplasia after an extended latent period by an unknown
mechanism. The U3 region of the LTR identified here as important in
trans-activation of the proto-oncogene c-jun and the
transcription factor complex AP-1 has been recently shown to be a
critical determinant of leukemogenicity and latency of
Mo-MuLV
(5, 6, 7, 8, 9) . The
ability of this portion of the Mo-MuLV to regulate genes and
transcription factors associated with growth control and transformation
may be relevant to the oncogenic properties of the virus. These
findings suggest a novel mechanism of retrovirus-induced activation of
cellular gene expression, potentially contributing to leukemogenesis.
Furthermore, the ability of the retroviral LTR to regulate cellular
genes should be taken into consideration in the use of retroviral
vectors for genetic therapies.
We thank Drs. C. Cepko, R. Bravo, and T. Rothstein for
generously providing reagents.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)
is a type C leukemogenic retrovirus which causes T
lymphocyte leukemia or lymphoma in mice over a period of months. The
mechanism whereby Mo-MuLV causes T lymphocyte leukemia or lymphoma
after this long latent period is not well understood. Because the
leukemia viruses do not encode oncogenes which can directly transform
cells, one potential mechanism of MuLV leukemogenesis may be through
transcriptional activation of cellular genes, including
proto-oncogenes. We have previously reported that infection of murine
fibroblasts or human T lymphocyte lines with Mo-MuLV results in
up-regulation of a number of genes encoding cell surface antigens and T
cell activation antigens, including CD2, CD3, CD4, the T-cell receptor
chain and MHC class I antigen expression (1-4). The ability
of Mo-MuLV to trans-activate cellular genes is determined by a
noncoding region of the genome, the long terminal repeat (LTR), and is
localized to a region of the LTR which has been shown to be important
for
pathogenicity
(5, 6, 7, 8, 9) .
Stable transfection of cells with a single-copy retroviral LTR
trans-activated specific endogenous cellular genes and
specific transiently expressed reporter genes (3),
eliminating the possibility that trans-activation is due
to titration of a putative negative regulatory factor by multiple
copies of the LTR DNA. The trans-activation of specific
cellular genes by the Mo-MuLV LTR is independent of the physical
location of either element, occurring regardless of whether the LTR is
integrated (endogenous) or transiently expressed (exogenous), or
whether the responsive cellular gene is integrated or transiently
expressed.
Cell Culture and Reagents
Balb/c-3T3 cells and
HeLa cells were obtained from American Type Culture Collection. The
mouse fibroblast Balb/c-3T3 cell line and the Moloney MuLV-infected
Balb/c-3T3 cell line MoBalb
(13) were carried in
Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated donor bovine calf serum (Sigma), 2 mML-glutamine, 100 units of penicillin/ml, 100 mg of
streptomycin/ml. The human cervical carcinoma cell line HeLa was grown
in Dulbecco's modified Eagle's medium supplemented with 10%
heat-inactivated newborn calf serum (Sigma). HeLa cells stably infected
with the pZip-Neo-SV(X) vector were maintained in Dulbecco's
modified Eagle's medium supplemented with 10% heat-inactivated
newborn calf serum, 0.5 mg of Geneticin/ml, and glutamine plus
antibiotics as above. Geneticin and TPA were obtained from Sigma.
Poly(dI-dC), acetyl-CoA, staurosporine, and okadaic acid were obtained
from Sigma. [C]Chloramphenicol was obtained from
DuPont NEN.
RNA Blot Analysis
Total cellular RNA was isolated
by guanidine thiocyanate/phenol RNA extraction
(13) , quantified,
separated by electrophoresis on formaldehyde-agarose gels, and
transferred to BioBlot-supported nitrocellulose (Costar, Cambridge,
MA). Hybridizations were carried out at 42 °C in 50% formamide for
18 h. The filters were washed sequentially in 2 SSC, 0.5% SDS
and in 0.2
SSC, 0.1% SDS, at room temperature and at 42 °C,
respectively. Autoradiograms were scanned for quantitation, using an
LKB laser densitometer (Pharmacia Biotech Inc.). [
-
P]-Labeled DNA
Probes-Purified fragments of c-jun, c-fos,
MCP-1, collagenase IV, and
-actin cDNA were radiolabeled with
[
-
P]dCTP using random primer
synthesis
(14) . Murine
-actin, c-fos, and MCP-1
(JE) DNA probes have been described previously
(15) .
The c-jun probe was a 1.7-kb PstI fragment of murine
c-jun, The c-fos fragment was a 1.1-kb
EcoRI/SalI fragment of murine c-fos, and the
MCP-1 probe was a 0.75-kb fragment of murine MCP-1 (JE). The
collagenase IV probe was a 2.2-kb EcoRI fragment purchased
from ATCC, and the actin probe was a 0.7-kb PstI fragment of
murine
-actin. The collagenase IV RNA probe was made from pGEM T7
promoter using T7 RNA polymerase (Promega).
Transfection and Infection of Cells
Transient
transfections were performed using the DEAE-dextran method
(16) .
Cells were seeded in 60-mm dishes at 3 10
the day
before transfection. Five µg of CAT-containing reporter plasmids
were transfected together with 5 µg of LTR vector or control
plasmid. 48 h after transfection, cells were stimulated with TPA at 25
ng/ml, and harvested for CAT assay 2 h later. Total cell extracts were
made by the freeze-thaw method and the supernatants were used for CAT
assay. The CAT activity was quantitated in extracts as
described
(17) . Co-transfection with the
pRSV-
-galactosidase plasmid (0.5 µg/transfection) and
cytofluorometric analysis of
-galactosidase activity were
performed to normalize for the transfection efficiency, as
described
(3) . pZip-Neo-SV(X) is a vector developed and provided
by C. Cepko (Harvard Medical School, Boston, MA), which contains the 5`
LTR of Mo-MuLV and 5` genomic sequences up to base 739 (using the
nucleotide numbering system of Shinnick
(18) ), the 3` region of
Mo-MuLV from bases 7200 and including the 3` LTR, and also Mo-MuLV
bases from 5407 to 5766. In addition, this vector also contains the
prokaryotic gene from transposon Tn5, which confers resistance to
neomycin, the pBR322 origin of replication, and the SV40 origin of
replication. For stable transfections, the retroviral vector
pZip-Neo-SV(X) was transfected into a canine cell amphotropic packaging
line (DAMP) and filtered supernatants containing packaged vector were
used to infect Balb/c3T3 and HeLa cells. Neomycin-resistant colonies
were selected and cloned.
LTR Expression Vectors, CAT Reporter Genes, and PCR
Mutagenesis
LTR-containing vectors pML4, pXF-MoA, and pGUXDP
have been previously described and are shown in Fig. 1. pGUXDP
has a deletion of 27 (nucleotide 96-122) and 75 base pairs
(nucleotide 145-219) within the LTR U3 region (see Fig. 1).
The vector pCollCAT-73 was the generous gift of P. Angel
(19) .
The MCP-1 promoter-CAT genes were the generous gift of A. J. van der
Eb
(20) . The mutation of the AP-1-binding site in pCollCAT-73 to
create the plasmid pmutcollCAT-73 was performed using two synthetic
oligonucleotides of 18 bases in length, encompassing the
HindIII site on pCollCAT-73 to the XbaI site at
+74. The 5` primer contained a complete HindIII site, and
the 3` primer contained a complete XbaI site. The fragment
containing the mutation was generated by synthesizing two overlapping
fragments, denaturing and then annealing. Complementary strands were
synthesized from the overhangs of the two annealed fragments, and
ligation and transformation of bacteria were carried out. The mutation
was confirmed by chain termination DNA sequencing
(21) .
Figure 1:
Schematic drawings of the reporter
genes and the LTR expression vectors. pCollCAT-73 contains an intact
AP-1 site; pmutcollCAT-73 contains a mutated AP-1 site with a mutation
of TGAGTCA to TGAGTAC; pCollCAT-71 (not shown) contains a deletion of
the first two bases of the AP-1 binding site (TGAGTCA). Also depicted
are the various vectors derived from the Mo-MuLV provirus, including
pML4, pXFMoA, pMoV9, and pGUXDP (see Ref. 3 for description). The
open bar represents the LTR of Mo-MuLV and its flanking
proviral sequences, and the thin bar the adjacent cellular
sequences.
Electrophoretic Mobility Shift Assay
HeLa cell
nuclear extracts were isolated by the method of Andrews and
Faller
(22) . Binding reactions were performed for 20 min on ice
with 10 µg of total nuclear protein in 20 µl of 10 mM
Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 10%
glycerol, 3 µg of poly(dI-dC), 1 mM dithiothreitol, 1
nM phenylmethylsulfonyl fluoride, and 20,000 cpm of
P-labeled oligonucleotide labeled with T4 kinase
(Promega). The DNA-protein complexes were separated from the unbound
probe on a native 5% polyacrylamide gel. The gels were vacuum-dried and
exposed to Kodak X-AR film for 18 h. The sequence of the AP-1 consensus
binding site oligonucleotide was 5`-CGCTTGATGAGTCAGCCGGAA-3` and
3`-GCGAACTACTCAGTCGGCCTT-5`. The transversion mutant oligonucleotide
used for competition experiments was 5`-CGCTTGATGAGTACGCCGGAA-3` and
3`-GCGAACTACTCATGCGGCCTT-5`. The antibodies used in the EMSA,
polyclonal rabbit antisera against all Jun family members, JunB and
JunD, and polyclonal rabbit antibody against c-Fos, were purchased from
Santa Cruz Biotech (Santa Cruz, CA) or were the generous gifts of Dr.
Thomas Rothstein.
(
)
MCP-1 is known to be regulated by the transcription factor
AP-1. The expression of two other genes also inducible by AP-1,
collagenase IV, and c-jun, were also examined in MoBalb cells.
RNA blot analysis demonstrated that transcript levels for collagenase
IV were elevated 13-fold and transcript levels for c-jun were
elevated 15-fold in cells containing the Mo-MuLV genome (not shown in
Fig. 2
). We have previously shown that the entire leukemia virus
is not required for trans-activation of cellular genes,
including MCP-1
(3) . In order to examine whether the Mo-MuLV LTR
alone could activate collagenase IV and MCP-1, the packaged defective
retroviral vector pZip-Neo-SV(X), which contains two LTRs but no
structural viral sequences, was used to infect Balb cells. After
neomycin selection, the resulting cells had two copies of the Mo-MuLV
LTR stably integrated in their genomes. The expression of collagenase
IV mRNA was elevated 14-fold in Balb/c-3T3 cells with integrated
Mo-MuLV LTRs, compared to the expression of the control gene
-actin. Expression of MCP-1 mRNA was elevated 5-fold by the
presence of the Mo-MuLV LTR (Fig. 2). Stable expression of the
Mo-MuLV LTR into Balb cells resulted in over 60-fold increases in
c-jun mRNA (Fig. 3). Induction of specific cellular gene
transcripts by the Mo-MuLV LTR was not restricted to murine cells.
Integration of the Mo-MuLV LTR into HeLa cells resulted in a 14.7-fold
increase in c-jun mRNA levels (Fig. 3). In contrast, the
levels of other gene transcripts, including c-fos, did not
increase significantly in response to the Mo-MuLV LTR.
Figure 2:
Analysis of MCP-1 and collagenase IV
transcripts in Balb cells expressing Mo-MuLV or Mo-MuLV LTRs. A, total cellular RNA (20 µg) was obtained from Balb cells or
Balb cells infected with Mo-MuLV. Lane 1, Balb cells; lane
2, Balb cells infected with Mo-MuLV. B, total cellular
RNA (20 µg) was obtained from Balb cells or Balb cells stably
infected with pZip-Neo-SV(X). RNA was separated on a
formaldehyde-agarose gel, transferred to a nitrocellulose filter, and
hybridized with labeled collagenase IV, MCP-1, or -actin probes.
Lane 1, Balb cells; lane 2, Balb cells expressing
pZip-Neo-SV(X). The figures are autoradiograms. The migration positions
of the collagenase IV,
-actin, and MCP-1 transcripts are
indicated.
Figure 3:
RNA blot analysis of c-jun and
c-fos transcripts in Balb and HeLa cells expressing the
Mo-MuLV LTR. Total cellular RNA (20 µg) from Balb cells or HeLa
cells untreated or stably infected with pZip-Neo-SV(X) was separated on
a formaldehyde-agarose gel, transferred to a nitrocellulose filter, and
hybridized with labeled c-jun or c-fos probes, then
stripped and reprobed for -actin. Lane 1, RNA from
control Balb cells; lane 2, RNA from Balb cells containing an
integrated Mo-MuLV LTR; lane 3, RNA from control HeLa cells;
lane 4, RNA from HeLa cells containing an integrated Mo-MuLV
LTR. The figure is an autoradiogram. The positions of the
c-jun, c-fos, and
-actin transcripts are
indicated.
To better
study the effect of the Mo-MuLV LTR on trans-activation of
cellular gene promoters, a number of plasmids containing the Mo-MuLV
provirus (pMoV9), the Mo-MuLV LTR (pXF-MoA, pA5, pML4), or portions of
the LTR (pGUXDP) (Fig. 1) were introduced into Balb/c-3T3 cells
in combination with reporter gene constructs. Chimeric plasmids
containing 5`-flanking (regulatory) regions of the human collagenase IV
gene or the MCP-1 gene ligated the chloramphenicol transferase (CAT)
gene were used as reporters for LTR trans-activation. Plasmid
pCollCAT-73 contains the 7-base pair (-73 to -67)
TPA-responsive element (TRE), which constitutes a binding site for
transcription factor AP-1
(19, 23) . The plasmid
pMCPCAT-2600 contains 2500 base pairs of 5`-regulatory sequence,
including two putative AP-1 binding sites. The plasmid containing the
provirus pMoV9 and the CAT reporter plasmids pCollCAT-73 or
pMCPCAT-2600 were co-transfected into Balb/c-3T3 cells. The
co-transfection of pMoV9 and pCollCAT-73 resulted in nearly 50-fold
trans-activation of the CAT activity, and the co-transfection
of pMoV9 and pMCPCAT-2600 resulted in 33-fold trans-activation
of CAT activity (Fig. 4).
Figure 4:
Induction of the collagenase IV and the
MCP-1 gene promoters by pMoV9 in transient expression assays. Balb
cells were transiently transfected with pCollCAT-73 together with a
control plasmid pBR322 (lane 1), or with the plasmid pMoV9,
containing the Mo-MuLV provirus (lane 2). Balb cells were
transiently transfected with pMCPCAT-2600 together with a control
plasmid pBR322 (lane 3), or with the plasmid pMoV9 (lane
4). Cells were harvested and extracts were assayed for CAT
activity. The products were separated by thin layer chromatography, and
an autoradiogram of the plate is shown here. The migration positions of
chloramphenicol (Cam) and the acetylated products
(AcCam) are indicated.
The viral LTR alone was as potent as
the entire proviral genome in inducing trans-activation of the
collagenase promoter in transient expression assays. In experiments
using pCollCAT-73 as reporter gene, CAT activity was increased over
10-fold when the plasmid pML4, containing a single LTR, was
co-transfected (Fig. 5A). Reporter genes with mutations
in the AP-1 binding site were employed to determine if this binding
site was required for trans-activation by the viral LTR. The
vectors pCollCAT-71, in which the first two bases of the TRE consensus
site are deleted, and pmutcollCAT-73, which contains a transversion of
the last two bases of the binding site (CA to AC) were used as reporter
genes in co-transfections with the vector pML4. Co-transfection of the
viral LTR did not result in trans-activation of CAT activity
from these TRE-mutant promoters (Fig. 5, A and
B).
Figure 5:
Regulation of collagenase IV promoter
mutants by a co-transfected Mo-MuLV LTR. A, Balb cells were
transiently transfected with pCollCAT-60 alone (lane 1), or
pCollCAT-60 together with pML4 (lane 2), with pCollCAT-71
alone (lane 3), or pCollCAT-71 together with pML4 (lane
4), with pCollCAT-73 alone (lane 5), or pCollCAT-73
together with pML4 (lane 6) for 48 h prior to harvesting and
assay for CAT activity. B, Balb cells were transiently
transfected with pCollCAT-73 alone (lane 1), or pCollCAT-73
together with pXFMoA (lane 2), with pmutcollCAT73 alone
(lane 3), or pmutcollCAT73 together with pXFMoA (lane
4) for 48 h prior to harvesting and assay for CAT activity. The
products were separated by thin layer chromatography, and
autoradiograms of the plates are shown here. The migration positions of
chloramphenicol (Cam) and the acetylated products
(AcCam) are indicated.
These experiments demonstrated that the TRE consensus
site, which serves as binding site for transcription factor AP-1, was
required for trans-activation by the LTR. To determine whether
the presence of the LTR increased AP-1 binding activity in the cell,
EMSAs for AP-1 DNA binding activity were performed using proteins from
HeLa cell nuclear extracts incubated with a radiolabeled TRE consensus
oligo. HeLa cell lines stably infected with a deleted retroviral vector
containing two Mo-MuLV LTRs were also used for preparation of nuclear
proteins. Cellular transcription factor AP-1 DNA binding activity was
greatly augmented in the cells containing stably integrated Mo-MuLV
LTRs (Fig. 6, lanes 1 and 2). To demonstrate
that this DNA binding activity to the TRE consensus oligo was specific,
a 100-fold excess of unlabeled TRE consensus or mutant oligo was
incubated with the nuclear extracts. The wild-type AP-1 binding
site-containing oligo competed efficiently for binding activity
(lanes 3 and 6), while the same amount of unlabeled
mutated TRE-site oligo (CA to AC transversion) could not compete away
the binding (lanes 4 and 7). As a control, TPA, a
known activator of AP-1 DNA binding activity, was used to stimulate the
HeLa cells (lane 5). The DNA binding complexes stimulated by
both TPA and Mo-MuLV LTR showed similar patterns on EMSA, both
complexes could be competed away by unlabeled wild-type AP-1 oligos,
and neither of the complexes were competed away by incubation with the
cold TRE-mutant oligo containing the CA to AC transversion (lanes 4 and 7).
Figure 6:
Induction of AP-1 activity by the Mo-MuLV
LTR. A radiolabeled oligonucleotide containing a TRE consensus sequence
was incubated with nuclear extracts made from: control HeLa cells
(lane 1); HeLa cells containing an integrated Mo-MuLV LTR
(pZip-Neo-SV(X)) (lane 2); HeLa cells containing an integrated
Mo-MuLV LTR (pZip-Neo-SV(X)) with an excess of cold TRE oligo added
during the binding reaction (lane 3); HeLa cells containing an
integrated Mo-MuLV LTR (pZip-Neo-SV(X)) with an excess of cold mutant
TRE oligo (CA to AC in the consensus sequence) added during the binding
reaction (lane 4); HeLa cells treated with 20 nM TPA
for 4 h (lane 5); HeLa cells treated with TPA, with an excess
of cold TRE oligo added during the binding reaction (lane 6);
HeLa cells treated with TPA, with an excess of cold mutant TRE oligo
(CA to AC in the consensus sequence) added during the binding reaction
(lane 7). The complexes were separated and analyzed as
described. The figure is an autoradiogram. The positions of the AP-1
complex and the free probe are indicated.
Because c-jun transcription was
strongly induced by the LTR in the absence of increases in c-fos transcription, the components of the AP-1 complex induced by the
LTR was investigated. Antibodies against Fos and Jun were preincubated
with the nuclear extracts to examine whether the binding of the AP-1
complex in the nuclear extracts to the TRE oligo could be interfered
with by the presence of the antibodies. The LTR-induced AP-1 DNA
binding to the TRE probe was almost completely inhibited by the
anti-pan-Jun antibody, while preincubation of the nuclear extracts with
anti-JunB or anti-JunD did not result in any interference with AP-1
binding to the TRE-specific probe, suggesting that the LTR-induced AP-1
DNA binding complex contained c-Jun protein (Fig. 7, A and B). The ability of the anti-JunB or anti-JunD
antibodies to interfere with the ability of their respective ligands to
participate in AP-1 complex formation was demonstrated by their
inhibition of AP-1 complex formation induced by TPA treatment
(Fig. 7B). Preincubation of a polyclonal antibody
against c-Fos with nuclear extracts from both HeLa cells treated with
TPA and HeLa cells containing the stably integrated Mo-MuLV LTR induced
a supershift on EMSA, indicating that c-Fos was also a component of the
LTR-induced DNA binding complex (Fig. 7C).
Figure 7:
Identification of the components of the
AP-1 complex induced by the LTR. A, a radiolabeled
oligonucleotide containing a TRE consensus sequence was incubated with
nuclear extracts made from: HeLa cells containing an integrated Mo-MuLV
LTR (pZip-Neo-SV(X)) (lane 1); control HeLa cells (lane
2); HeLa cells treated with 20 nM TPA for 4 h (lane
3); HeLa cells treated with 20 nM TPA for 4 h, with 1
µg of anti-pan-Jun antibody added for 15 min prior to the binding
reaction (lane 4); HeLa cells containing an integrated Mo-MuLV
LTR (pZip-Neo-SV(X)), with 1 µg of anti-pan-Jun antibody added for
15 min prior to the binding reaction (lane 5). B, a
radiolabeled oligonucleotide containing a TRE consensus sequence was
incubated with nuclear extracts made from: HeLa cells treated with 20
nM TPA for 4 h (lane 1); HeLa cells containing an
integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 2); HeLa cells
treated with 20 nM TPA for 4 h with 1 µg of anti-JunB
antibody added during the binding reaction (lane 3); HeLa
cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) with 1
µg of anti-JunB antibody added during the binding reaction
(lane 4); HeLa cells treated with 20 nM TPA for 4 h
with 1 µg of anti-JunD antibody added during the binding reaction
(lane 5); HeLa cells containing an integrated Mo-MuLV LTR
(pZip-Neo-SV(X)) with 1 µg of anti-JunD antibody added during the
binding reaction (lane 6). Quantitation of AP-1 complexes by
densitometer disclosed that whereas the presence of anti-JunB and
anti-JunD antibodies decreased TPA-induced complex formation by 43%,
there was no such inhibitory effect of the antibodies on the AP-1
complexes induced by the Mo-MuLV-LTR. C, a radiolabeled
oligonucleotide containing a TRE consensus sequence was incubated with
nuclear extracts made from: HeLa cells treated with 20 nM TPA
for 4 h (lane 1); control HeLa cells (lane 2); HeLa
cells containing an integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane
3); HeLa cells containing an integrated Mo-MuLV LTR
(pZip-Neo-SV(X)), with 1 µg of anti-c-Fos antibody added for 15 min
prior to the binding reaction (lane 4); HeLa cells treated
with 20 nM TPA for 4 h, with 1 µg of anti-c-Fos antibody
added for 15 min prior to the binding reaction (lane 5). The
complexes were separated and analyzed as described. The figures are
autoradiograms. The positions of the AP-1 complex and the free probe
are indicated.
We have
previously demonstrated that mutations of the LTR which prevent the
generation of a polymerase III transcript from the LTR render it
incapable of trans-activation of cellular genes (3). To determine the specificity of the induction of AP-1 activity by
the Mo-MuLV LTR, wild-type and mutant LTRs were co-transfected into
HeLa cells. Nuclear extracts were made from these transient
transfectants and then tested for their ability to enhance AP-1 DNA
binding activity. pXF-MoA, which contains the wild-type LTR, has been
shown to strongly activate a co-transfected pCollCAT-73 vector
(3).
Plasmid pGUXDP, which contains an LTR with a mutation
within the U3 region and does not generate a transcript, has been shown
to have no trans-activating ability for MHC or MCP-1 genes
(3). Nuclear extract made from pXF-MoA-transfected HeLa cells showed a
5-fold induction of AP-1 DNA binding activity, in comparison to
extracts from control HeLa cells (Fig. 8). Nuclear extracts made
from pGUXDP transfectants, however, demonstrated no
trans-activation of AP-1 DNA binding activity. Thus, the
trans-activation ability of the LTR correlates with the
ability to activate AP-1 DNA binding activity.
Figure 8:
Induction of AP-1 activity by vectors
containing deleted LTRs. A radiolabeled oligonucleotide containing a
TRE consensus sequence was incubated with nuclear extracts made from:
control HeLa cells (lane 1); HeLa cells transiently
transfected with pGUXDP (lane 2); HeLa cells transiently
transfected with pXFMoA (lane 3); HeLa cells treated with 20
nM TPA for 4 h (lane 4). The complexes were separated
and analyzed as described. The figures are autoradiograms. The
positions of the AP-1 complex and the free probe are
indicated.
The expression of
integrated, cellular AP-1 inducible genes could be induced by even
transient expression of the Mo-MuLV LTR. Collagenase IV mRNA was
elevated 8-fold in Balb cells transiently transfected with pMoV9,
compared to cells transfected with a control plasmid not containing an
LTR. The expression of the -actin gene was unaffected
(Fig. 9, lanes 1 and 2). As a control,
treatment with TPA was also used to stimulate collagenase IV mRNA
expression (Fig. 9, lane 3). These findings demonstrate
again that induction of the expression of specific cellular genes by
the MoMuLV LTR is independent of the location of either the LTR or the
responsive gene.
Figure 9:
RNA blot analysis of collagenase IV
transcripts in Balb cells transiently transfected with pMoV9. Total
cellular RNA (20 µg) from Balb cells or Balb cells transiently
transfected pMoV9 were separated on a agarose gel, transferred to a
nitrocellulose filter, and hybridized with a radiolabeled collagenase
RNA probe and subsequently with a probe for -actin. Lane
1, Balb cells; lane 2, Balb cells transiently transfected
with pMoV9; lane 3, Balb cells stimulated with TPA for 2 h.
The figures are autoradiograms. The migration positions of the
collagenase IV,
-actin, and MCP-1 transcripts are
indicated.
The protein kinase C inhibitor staurosporine and
the protein serine/threonine phosphatase inhibitor okadaic acid have
been reported to affect TPA induction of AP-1 DNA binding activity. To
study the effect of staurosporine or okadaic acid on LTR induction of
AP-1 activity, electrophoretic mobility shift assays for AP-1 binding
activity were performed using HeLa cell nuclear extract incubated with
a radiolabeled AP-1 consensus oligo. HeLa cell lines containing a
stably integrated Mo-MuLV LTR (pZip-Neo-SV(X)) were also used for
preparation of nuclear proteins. As expected, AP-1 DNA binding activity
was greatly augmented in the cells containing an integrated Mo-MuLV
LTR. After treatment with staurosporine for 6 h, however, the induction
of AP-1 DNA binding activity by the LTR was reduced by 50%
(Fig. 10A, lanes 2 and 3). Basal expression of
the AP-1 complex in control cells not expressing the LTR was not
affected by staurosporine (not shown). The TPA induction of AP-1 DNA
binding activity was also reduced by 50% after the same treatment with
staurosporine (Fig. 10A, lanes 4 and 5). In
contrast, the protein phosphatase inhibitor okadaic acid increased
basal expression and the enhanced induction of AP-1 DNA binding
activity by the Mo-MuLV LTR (Fig. 10B, lanes 1 and
2).
Figure 10:
The effect of protein kinase C inhibitors
and serine/threonine phosphatase inhibitors on Mo-MuLV LTR induction of
AP-1 activity. A, a radiolabeled oligonucleotide containing a
TRE consensus sequence was incubated with nuclear extracts made from:
control HeLa cells (lane 1); HeLa cells with a stably
integrated Mo-MuLV LTR (pZip-Neo-SV(X)) (lane 2); HeLa cells
with a stably integrated Mo-MuLV LTR treated with staurosporine at 0.1
µM for 6 h (lane 3); HeLa cells stimulated with
TPA for 6 h (lane 4); HeLa cells stimulated with TPA and
treated with staurosporine at 0.1 µM for 6 h (lane
5). B, HeLa cells (lane 1); HeLa cells were
treated with okadaic acid at 0.1 µM for 6 h (lane
2); HeLa cells with a stably integrated Mo-MuLV LTR
(pZip-Neo-SV(X)) (lane 3); HeLa cells with stably integrated
Mo-MuLV LTR treated with okadaic acid at 0.1 µM for 6 h
(lane 4). The complexes were separated and analyzed as
described. The figures are autoradiograms. The positions of the AP-1
complex and the free probe are indicated.
The studies reported
herein confirm and extend these findings to another group of cellular
genes, and define a transcription complex and cis-acting
promoter element required for this trans-activation.
Trans-activation of the distinct set of
12-O-tetradecanoylphorbol-13-acetate (TPA)-inducible genes by
the Mo-MuLV LTR reported here is independent of the physical location
of the genes or of the LTR, occurring whether the LTR is integrated in
the genome or transiently expressed, and whether the responsive
cellular genes or gene promoters are integrated or transiently
expressed.
When pGUXDP was co-transfected with a collagenase promoter-CAT
vector, both the trans-activation of CAT activity and the
activation of cellular AP-1 DNA binding activity were minimal compared
to the effects of the wild-type LTR (e.g. pXFMoA).
(
)
but we have no further evidence that redox mechanisms
regulate transcription of AP-1 inducible genes or the formation of AP-1
binding complexes by the viral LTR.
(
)
Alternatively, the activation of c-jun and AP-1 may indirectly affect the expression of these genes.
Overexpression of c-jun in malignant cells has been reported
to affect MHC class II expression, and transfection of c-jun and c-fos genes into B-16 melanoma cells resulted in the
activation of H-2 class I gene expression, suggesting that the
activation of other genes which are not directly regulated by AP-1 may
be affected indirectly
(49) .
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.