Department of Surgery, University of Maryland Medical School and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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Activator protein 1 (AP-1) is a group of dimeric
transcription factors composed of protooncogene (Jun and Fos) subunits
that bind to a common DNA site, the AP-1 binding site. The proteins of
c-Jun, JunB, and Fos are essential for initiation of the cell cycle.
Conversely, the activation of the junD
gene slows cell growth in some cell types. The current study tests the
hypothesis that polyamines influence cell growth by altering the
balance of positive and negative Jun/AP-1 activities in intestinal
epithelial cells. Studies were conducted in the IEC-6 cell line derived
from rat small intestinal crypt cells. Administration of
-difluoromethylornithine (DFMO), a specific inhibitor for polyamine
synthesis, for 4 and 6 days completely depleted cellular polyamine
levels, while AP-1 binding activity was significantly increased.
Spermidine, when given together with DFMO, restored AP-1 binding
activity toward normal. The increased AP-1 complexes in
polyamine-deficient cells were dramatically supershifted by the
anti-JunD antibody but not by antibodies against c-Jun, JunB, or Fos
proteins. There were significant increases in JunD mRNA and protein in
DFMO-treated cells, although expression of the
c-fos,
c-jun, and
junB genes decreased. The increase in
JunD/AP-1 activity in DFMO-treated cells was associated with a
significant decrease in cell division. Exposure of control quiescent
cells to 5% dialyzed serum increased c-Jun/AP-1 but not JunD/AP-1
activities. DFMO prevented the stimulation of c-Jun/AP-1 activity
induced by 5% dialyzed serum. These results indicate that
1) polyamine depletion is associated
with an increase in AP-1 binding activity and
2) the increase in AP-1 activity in
the DFMO-treated cells was primarily contributed by an increase in the
JunD/AP-1. These findings suggest that polyamines regulate cell growth
at least partially by modulating the balance of positive and negative
Jun/AP-1 activities in the intestinal mucosa.
protooncogenes; ornithine decarboxylase; proliferation; mucosal growth
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INTRODUCTION |
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THE TRANSCRIPTION FACTOR activator protein 1 (AP-1) has been shown to integrate various mitogenic signals in a large number of cell types and plays a critical role in the control of cell proliferation (2). AP-1 is a homo- or heterodimeric DNA-binding protein composed of either two Jun family proteins or one Jun and one Fos family protein via the interaction of the leucine zipper domains (1, 2). The activated AP-1 dimer specifically binds to DNA at a consensus AP-1 site (TGAC/GTCA) in the responsive regions of gene promoter and regulates the transcriptional activity of AP-1-dependent genes (2, 26, 27). Although Jun-Fos heterdimers are more stable and more potent at binding DNA compared with corresponding Jun-Jun dimers, Fos proteins alone do not form stable Fos-Fos homodimers and do not bind DNA without Jun participation. Therefore, Jun proteins are more important than Fos proteins in gene regulation because they are absolutely required for the functioning of the AP-1 transcription factors (2, 30).
Numerous studies have shown that c-jun and junB function as immediate early response genes and are involved in the transition from a quiescent state to a proliferating state after exposure to growth factors (2, 4, 10). Inhibition of expression of the c-jun and junB genes by antisense RNA or oligomers causes reversible growth arrest and withdrawal from the cell cycle (28), indicating that c-Jun and JunB are positive AP-1 factors for cell proliferation. In contrast, the activation of junD expression results in slower cell growth and an increase in the percentage of cells in the G0/G1 phase of the cell cycle (11, 22, 25). It has been shown that the inhibition of proliferation by 1,25-dihydroxyvitamin D3 in chronic myelogenous leukemia cells is accompanied by an increase in AP-1 DNA binding activity and that JunD protein is the major constituent of these AP-1 complexes (14). The activation of junD expression also partially suppresses neoplastic transformation induced by an activated ras oncogene (11). These findings clearly indicate that JunD is a negative AP-1 factor that downregulates cell proliferation in some cell types.
A series of observations from our previous studies (32, 33, 35) and
others (12, 15, 16) has demonstrated that the polyamines spermidine and
spermine and their diamine precursor putrescine are absolutely required
for cell proliferation in the crypts of the small intestinal mucosa.
The intracellular concentration of polyamines is highly regulated by
the cell according to the state of growth (12) and completely depends
on the activation or inhibition of ornithine decarboxylase (ODC), the
first rate-limiting step in polyamine biosynthesis (17). Decreasing
cellular polyamine levels by inhibition of the activity of ODC with
-difluoromethylornithine (DFMO) suppresses cell renewal in
intestinal mucosal tissue (16, 35). Although the exact role of
polyamines in the regulation of cell proliferation is still unclear, we
have recently demonstrated that polyamine deficiency results in a
significant decrease in expression of the
c-fos,
c-myc, and
c-jun genes in intestinal crypt cells
(36). To further investigate the molecular mechanism of mucosal growth
inhibition following polyamine depletion, the current studies were
designed to determine whether polyamines alter the balance of positive
and negative Jun/AP-1 binding activities in intestinal epithelial cells
and whether depletion of intracellular polyamines by DFMO results in an
increase in the negative AP-1 factor JunD. Some of these data have been
published in abstract form (18).
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MATERIALS AND METHODS |
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Cell culture and general experiment protocol. The IEC-6 cell line was purchased from the American Type Culture Collection (ATCC) at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (23). Originating from intestinal crypt cells as judged by morphological and immunologic criteria, the IEC-6 cells are nontumorigenic and retain the undifferentiated character of epithelial stem cells.
Stock cells were maintained in T-150 flasks in DMEM supplemented with 5% heat-inactivated FBS, 10 µg/ml insulin, and 50 µg gentamicin sulfate/ml. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once per week at 1:20, and medium was changed three times weekly. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative. Passages 15-20 were used in the experiments. There were no significant changes of biological function and characterization from passages 15-20.
The general protocol of the experiments and the methods used were similar to those described previously (20, 36). In brief, IEC-6 cells were plated at 6.25 × 104 cells/cm2 in DMEM + 5% dialyzed FBS + 10 µg insulin and 50 µg gentamicin sulfate/ml (supplemented DMEM). The cells were incubated in a humidified atmosphere at 37°C in 90% by volume of air and 10% by volume of CO2 for 24 h before being subjected to a series of experimental treatments.
In the first series of studies, we examined whether cellular polyamines could alter AP-1 DNA binding activity in IEC-6 cells. Cells were grown in control cultures or cultures containing either DFMO (5 mM) or DFMO plus 5 µM spermidine for 4 and 6 days. The dishes were placed on ice, the monolayers were washed three times with ice-cold Dulbecco's phosphate-buffered saline (DPBS), and then nuclear proteins were prepared. The AP-1 DNA binding activity was measured by electrophoretic mobility shift assay. To characterize the AP-1 complex in polyamine-deficient cells, the compositional changes of the AP-1 complex were examined by gel supershift assays using various antibodies against either c-Fos, c-Jun, JunB, or JunD proteins.
In the second series of studies, we investigated the effect of polyamine depletion by DFMO on the expression of protooncogene junD in IEC-6 cells. After cells were grown in the presence or absence of DFMO for 4 and 6 days after initial plating, JunD mRNA and protein levels were measured by Northern and Western blotting analyses. The cellular distribution of JunD protein in control and polyamine-deficient cells was also examined by immunohistochemical staining.
Preparation of nuclear protein extraction and
electrophoretic mobility shift assay. Nuclear extracts
were prepared as previously described (38). Briefly, cells were
harvested in ice-cold DPBS with a cell scraper and centrifuged at 500 g at 4°C for 5 min. The resulting
cell pellets were resuspended in 4 ml of ice-cold STM buffer containing
20 mM Tris · HCl (pH 7.85), 250 mM sucrose, 1.1 mM
MgCl2, and 0.2% Triton X-100 and
were incubated on ice for 5 min. The cell pellets were then washed once
with STM buffer containing 0.2% Triton X-100 and once with STM buffer
without Triton X-100. The isolated nuclei were then resuspended in STM buffer (without Triton X-100) that contained 0.4 M KCl and 5 mM -mercaptoethanol and incubated on ice for 10 min to extract nuclear proteins. After centrifugation at 2,000 g at 4°C for 10 min, the supernatant was collected, aliquoted, and frozen at
80°C
before use. The protein content of nuclear extracts was determined by the method described by Bradford (5).
The double-stranded oligonucleotides used in these experiments
included
5'-CGCTTGGCCGGAA-3',
which contains a consensus AP-1 binding site that is underlined
(Santa Cruz Biotechnology, Santa Cruz, CA). These oligonucleotides were
radioactively end-labeled with
[
-32P]ATP (3,000 Ci/mmol; Amersham, Arlington Heights, IL) and T4 polynucleotide kinase
(Promega, Madison, WI). For mobility shift assays, 0.035 pmol
32P-labeled oligonucleotides
(~30,000 cpm) and 10 µg nuclear protein (in 2 µl) were incubated
in a total volume of 25 µl in the presence of 2 mM
Tris · HCl (pH 7.5), 8 mM NaCl, 0.2 mM EDTA, 0.2 mM
-mercaptoethanol, 0.8% glycerol, and 1 µg poly(dI-dC). The
binding reactions were allowed to proceed at room temperature for 20 min. Thereafter, 2 µl of bromophenol blue (0.1% in water) were
added, and protein-DNA complexes were resolved by electrophoresis on
nondenaturing 5% polyacrylamide gels and were visualized by autoradiography.
Gel supershift assays were accomplished by adding 1 µg (in 1 µl) of TransCruz supershift antibody to the reaction mixture and incubating for an additional 30 min at room temperature. The antibodies used in these experiments included rabbit polyclonal IgG raised against either c-Jun, JunB, or JunD, and a polyclonal antibody raised against all Fos proteins including c-Fos, FosB, Fra-1, and Fra-2. All gel supershift antibodies were purchased from Santa Cruz Biotechnology.
RNA isolation and Northern blot
analysis. Total RNA was extracted with guanidinium
isothiocyanate solution and purified by CsCl density gradient
ultracentrifugation as described by Chirgwin et al. (8). Briefly, the
monolayer of cells was washed with DPBS and lysed in 4 M guanidinium
isothiocyanate. The lysates were brought to 2.4 M CsCl concentration
and centrifuged through a 5.7-M CsCl cushion at 150,000 g at 20°C for 24 h. After
centrifugation, the supernatant was aspirated and the tube was cut
about 0.5 cm from the bottom with a flamed scalpel. The resulting RNA
pellet was dissolved in Tris · HCl (pH 7.5)
containing 1 mM EDTA, 5% sodium laurylsarcosine, and 5% phenol (added
just before use). The purified RNA was precipitated from the aqueous
phase by the addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of
ethanol in sequence. The final RNA was dissolved in water and estimated from its ultraviolet absorbance at 260 nm using a conversion factor of
40 units. In most cases, 30 µg of total cellular RNA were denatured and fractionated electrophoretically using a 1.2% agarose gel containing 3% formaldehyde and was transferred by blotting to nitrocellulose filters. Blots were prehybridized for 24 h at 42°C with 5× Denhardt's solution and 5× standard salmon sperm
DNA. DNA probes for JunD (Oncogene Science) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH, ATCC 57090) were
labeled with
[-32P]dCTP using a
standard nick translation procedure. Hybridization was carried out
overnight at 42°C in the same solution containing 10% dextran
sulfate and 32P-labeled DNA
probes. Blots were washed with two changes of 1× saline-sodium
citrate-0.1% SDS for 10 min at room temperature. After the final wash,
the filters were autoradiographed with intensifying screens at
70°C. The signals were quantitated by densitometry analysis
of the autoradiograms.
Western blot analysis of Jun and Fos proteins. Nuclear extracts were prepared as described above. Ten micrograms of nuclear protein were mixed with SDS sample buffer, boiled for 10 min, and then subjected to electrophoresis on 7.5% acrylamide gels according to Laemmli (13). Briefly, after the transfer of protein onto nitrocellulose filters, the filters were incubated for 1 h in 5% nonfat dry milk in 10× phosphate-buffered saline-Tween 20 [PBS-T: 15 mM NaH2PO4, 80 mM Na2HPO4, 1.5 M NaCl, pH 7.5, and 0.5% (vol/vol) Tween 20]. Immunologic evaluation was then performed for 1 h in 1% BSA-PBS-T buffer containing 1 µg/ml polyclonal antibodies that had been used for gel supershift assays as described above. The filters were subsequently washed with 1× PBS-T and incubated for 1 h with goat anti-rabbit IgG antibody conjugated to peroxidase. After extensive washing with 1× PBS-T, the immunocomplexes on the filters were reacted for 1 min with chemiluminescence reagent (NEL-100, DuPont NEN). Finally, the filters were placed in a plastic sheet protector and exposed to autoradiography film for 30 or 60 s.
Immunohistochemical staining. Immunohistochemical staining for JunD protein was performed in IEC-6 cells by the indirect immunoperoxidase method as described previously (37). Cells were plated at 4.0 × 104 cells/cm2 on 22 × 22-mm glass coverslips that were placed in 35-mm dishes in medium consisting of DMEM + 5% dialyzed FBS + 10 µg/ml insulin and 50 µg/ml gentamicin sulfate. DFMO (5 mM) with or without 5 µM spermidine was added as treatment. On day 6 after initial plating, the cells were washed with DPBS and then with DPBS without calcium and magnesium (DPBS-Ca2+-Mg2+) and were fixed for 5 min at room temperature in 1 part 37% paraformaldehyde + 9 parts PEM buffer (10 mM PIPES, 5 mM EGTA, 2 mM MgCl2, pH 6.8, containing 0.2% Triton X-100). The cells were postfixed for 5 min with ice-cold methanol. The cells were rehydrated in DPBS-Ca2+-Mg2+ for 30 min at room temperature and were incubated with 5% goat serum for 10 min at room temperature to reduce nonspecific background staining. The cells were then incubated with rabbit polyclonal anti-JunD antibody used for gel supershift assays as described above at a dilution of 1:1,000 in humidified chambers for 20 min at room temperature. The primary antibody recognizes the 40-kDa JunD protein band in immunoblots of IEC-6 cell extracts and does not cross-react with other oncoproteins. Nonspecific slides were incubated without antibody to JunD protein. All the treated slides were incubated for 20 min with second antibody-horseradish peroxidase conjugate at a dilution of 1:500. The final reaction was achieved by incubating with freshly prepared reagent containing 3-amino-9-ethylcarbazole (Sigma) dissolved in dimethylformamide and sodium acetate. The slides were counterstained with hematoxylin, mounted, and viewed with an Olympus microscope.
Measurement of DNA synthesis. DNA synthesis was measured with the use of the [3H]thymidine incorporation technique as previously described (29). Cells in 24-well plates were pulsed with 1 µCi/ml tritiated thymidine for 4 h before harvest. Cells were washed twice with cold DPBS solution and then incubated in cold 10% trichloroacetic acid (TCA) for 30 min at 4°C. After rinsing twice with cold 10% TCA, cells were dissolved in 0.5 ml of 0.5 N NaOH at 37°C in humidified air for 90 min. The incorporation of [3H]thymidine into DNA was determined by counting the aliquot of cell lysate in a Beckman liquid scintillation counter. The protein content of aliquot of cell lysate was determined by the method described by Bradford (5). DNA synthesis was expressed as disintegrations per minute per microgram of protein.
Polyamine analysis. The nuclear
polyamine content was analyzed by HPLC as described previously (36).
Nuclei were prepared according to the method described above, mixed
with 0.5 M perchloric acid, and then frozen at 80°C until
ready for extraction, dansylation, and HPLC. The standard curve
encompassed 0.31-10 µM. Values that fell more than 25% below
the curve were considered not detectable. Protein was determined by the
Bradford method (5). The results are expressed as nanomoles of
polyamines per milligram of protein.
Statistics. All data are expressed as means ± SE from six dishes. Autoradiographic results were repeated three times. The significance of the difference between means was determined by analysis of variance. The level of significance was determined using the Duncan's multiple range test (9).
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RESULTS |
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Effect of polyamine depletion on AP-1 DNA binding
activity. Exposure of IEC-6 cells to 5 mM DFMO for 4 or
6 days almost completely depleted cellular polyamines. The levels of
putrescine and spermidine were undetectable and spermine content was
decreased by ~60% on days 4 and
6 in the presence of DFMO (data not
shown). Similar results have been published previously (36). Putrescine
level was undetectable in nuclei isolated from both control and
DFMO-treated cells. Normal basal levels of nuclear spermidine and
spermine were slightly lower than those observed in whole cells.
Administration of DFMO markedly decreased nuclear spermidine and
spermine levels (Fig. 1). In cells exposed
to DFMO for 4 and 6 days, nuclear spermidine was undetectable and
spermine was decreased by ~70%.
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Depletion of nuclear polyamines by DFMO significantly increased AP-1
binding activity in IEC-6 cells as measured by electrophoretic mobility
shift assay (Fig. 2). The AP-1 binding
activity was increased by ~2.2-fold on day
4 and by ~3.1-fold on day
6 in the DFMO-treated cells, although the basal AP-1
binding activity in control cells (without DFMO) increased with time
after initial plating. Spermidine (5 µM) given together with DFMO
attenuated the increase in AP-1 binding activity at
day 4 and prevented the increase at day
6. Putrescine at a dose of 10 µM had
an effect equal to spermidine on the AP-1 binding activity when it was
added to cultures that contained DFMO (data not shown). In cells grown
in standard culture conditions (without DFMO), the AP-1 DNA binding
activity was not altered by addition of exogenous spermidine (5 µM)
itself (data not shown).
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To evaluate the specificity of the binding reactions in Fig. 2, the
competitive inhibition experiments were performed. As can be seen in
Fig. 3, AP-1 binding activities in control
cells and cells exposed to DFMO were dose-dependently inhibited when various concentrations of the unlabeled AP-1 oligonucleotide were added
to the binding reaction mixture. We also examined the effect of the
unlabeled oligonucleotide containing a mutated AP-1 binding site on
AP-1 binding activity and demonstrated that the AP-1 mutated oligonucleotide did not inhibit AP-1 binding activity (data not shown).
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Effect of polyamine depletion on AP-2 and SP1 binding
activities. The AP-2 DNA binding activity was
undetectable in IEC-6 cells regardless of the presence or absence of
DFMO for 4 and 6 days (Fig.
4A). As
shown in Fig. 4B, basal level of SP1
binding activity was high in normal IEC-6 cells (without DFMO).
Treatment with DFMO for 4 and 6 days significantly decreased SP1
binding activity, which was completely prevented by exogenous
spermidine. The competition experiments were also performed by using an
unlabeled oligonucleotide containing a single SP1 binding site. The
SP1 binding activity in IEC-6 cells was almost completely
inhibited when unlabeled SP1 oligonucleotide (×50) was
added to the binding reaction mixture (data not shown). Because
other transcription factors such as AP-2 showed no significant
difference between control cells and polyamine-deficient cells and
since SP1 binding activity decreased in DFMO-treated cells, the
increase in AP-1 binding activity observed following polyamine
depletion was specific.
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Characterization of AP-1 complexes in the
polyamine-deficient IEC-6 cells. Because AP-1 complexes
consist of Jun and Fos proteins, we next examined the compositional
changes of the induced AP-1 complexes that occur following polyamine
depletion. These studies were carried out by performing gel supershift
assays using various specific antibodies. As can be seen in Fig.
5, the anti-JunD antibody, when added to
the binding reaction mixture, dramatically supershifted the AP-1
complexes present in IEC-6 cells exposed to DFMO for 4 and 6 days. The
AP-1 activity attributed to JunD in the DFMO-treated cells was
approximately one-third of the total AP-1 binding activity on
day 4 and about one-half on
day 6. In control cells and cells exposed to DFMO and spermidine, the AP-1 binding activity was slightly
supershifted by the anti-JunD antibody. On the other hand, addition of
antibodies against c-Jun or JunB to the binding reaction mixture had
effect on the AP-1 binding activity in all three treatment
groups. There was no stimulation of the AP-1 activity attributed to
c-Jun or JunB by DFMO (Fig. 6,
A and
B). Although the anti-Fos antibody
also partially supershifted the AP-1 complexes, there were no
significant differences in the AP-1 activity attributed to Fos between
control cells and cells exposed to DFMO or DFMO plus spermidine (Fig.
6C). The increased AP-1 binding
activities in polyamine-deficient cells were not supershifted by the
anti-Myc IgG, which served as a control antibody. These results
indicate that the increase in AP-1 activity in polyamine-deficient
cells is primarily contributed by an increase in JunD/AP-1 while
c-Jun/AP-1 and JunB/AP-1 activity remains essentially unchanged.
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Changes in JunD mRNA and protein levels following
polyamine depletion. To determine whether the increased
JunD/AP-1 activity following polyamine depletion resulted from the
activation of junD gene expression, we
measured changes in the JunD mRNA and protein levels in the
DFMO-treated cells. Consistent with its effect on JunD/AP-1 binding
activity, inhibition of polyamine synthesis by DFMO resulted in a
significant increase in steady-state levels of JunD mRNA (Fig.
7). Increased JunD mRNA levels in the DFMO-treated cells were 6.6 times the control level on
day 4 and 8.9 times on
day 6. Addition of spermidine (5 µM)
completely prevented the stimulatory effects of DFMO on expression of
the junD gene. The induced mRNA levels
of JunD in cells treated with DFMO returned toward control levels when
spermidine was given. There were no changes in GAPDH mRNA levels in
IEC-6 cells grown in the presence or absence of DFMO.
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Western immunoblotting analysis of IEC-6 cells showed the JunD protein
at 40 kDa, the expected molecular mass of JunD in other epithelial
cells. Increased levels of JunD mRNA following polyamine depletion were
paralleled by an increase in JunD protein (Fig. 8). There were significant increases in
JunD protein levels of the nuclear extracts comparable to those used
for gel shift assays in IEC-6 cells exposed to DFMO for 4 and 6 days.
In the presence of DFMO, exogenous spermidine returned the induced
levels of JunD protein toward normal values.
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We also measured changes in the levels of mRNAs and proteins for c-fos, c-jun, and junB in cells treated with DFMO for 4 and 6 days and demonstrated that polyamine depletion was associated with significant decreases in expression of these genes (data not shown). Similar data have been published previously (20, 36).
Effects of polyamine deficiency on the distribution of JunD
protein in IEC-6 cells. To extend the positive findings
of increased JunD following polyamine depletion, immunohistochemical
staining was performed to determine the organization of JunD protein in IEC-6 cells. Figure 9 shows the cellular distribution of JunD protein
in cells grown in the presence of DFMO with or without spermidine for 6 days. In control cells, the slight immunostaining for JunD protein was
visible and present just inside the nuclei (Fig.
9A).
These results were consistent with our data from Northern and Western
blot analyses that revealed a basal level of expression of the
junD gene in control cells. In the
DFMO-treated cells (Fig. 9B),
however, these nuclear immunoreactivities for JunD markedly increased
as expected. Identification was facilitated for every experiment by
heavily staining nuclei. The appearance of JunD in the cells treated
with DFMO plus spermidine was the same as in control cells (Fig.
9C). The distribution and the
immunostaining level for JunD in cells grown in the presence of DFMO
and spermidine were indistinguishable from those of control cells.
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Association of increased JunD/AP-1 binding activity
with a decrease in cell division. To elucidate the
possible role of increased JunD/AP-1 binding activity in cell
proliferation, we measured changes in the rate of DNA synthesis and
final cell number in IEC-6 cells exposed to DFMO or DFMO plus
spermidine. As can be seen in Fig. 10,
increased JunD/AP-1 activity in the DFMO-treated cells was associated
with significant decreases in DNA synthesis and cell number. The
administration of spermidine completely prevented the effects of DFMO
on AP-1 binding activity and cell proliferation. The induced JunD/AP-1
activity and decreased cell proliferation in the DFMO-treated cells
returned toward control levels when spermidine was given. In the cells
grown in standard culture conditions containing no DFMO, exogenous
polyamines had no additional effect on cell division (data not shown).
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To support the possibility that JunD/AP-1 plays a negative role in
intestinal mucosal growth, we measured the changes in Jun/AP-1 binding
activities in quiescent cells exposed to a positive treatment of 5%
dialyzed FBS. In this study, cells were grown in the presence or
absence of 5 mM DFMO for 4 days and were then exposed to 5% dialyzed
FBS after 24-h serum deprivation. Figure
11 clearly shows that exposure of normal
quiescent cells (without DFMO) to 5% dialyzed FBS markedly increased
AP-1 binding activities. However, there was no significant increase in
AP-1 binding activity in DFMO-treated cells exposed to 5% dialyzed
FBS. The observations from supershift assays showed that increased AP-1
activities in normal quiescent cells exposed to 5% dialyzed serum
resulted primarily from the increase in c-Jun/AP-1 and JunB/AP-1,
whereas JunD/AP-1 activity in polyamine-deficient cells was not induced
by 5% dialyzed FBS. These results suggest that JunD/AP-1 appears to
function differently from c-Jun/AP-1 and JunB/AP-1 in regulating cell
growth in the intestinal mucosa.
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DISCUSSION |
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The murine junD was cloned in 1989 as a third member of the protooncogene jun family by screening cDNA libraries with c-jun and/or junB probes (11, 25). Unlike c-jun and junB, junD is usually constitutively expressed and is not inducible by growth factors (11, 14). Overexpression of the junD gene promotes neither cell proliferation nor transformation in several cell types (14, 22, 24), although some studies have shown a positive effect of junD on cell proliferation. Pfarr et al. (22) have reported that growth arrest correlates well with high JunD and low c-Jun levels while stimulation of cell proliferation is associated with low JunD and high c-Jun levels. Similar changes have also been observed in papillomavirus-transformed mouse cell lines in which an increase in the c-Jun- and JunB-to-JunD ratio becomes evident as cells progress from fibromatosis to fibrosarcoma (3). Recently, it has been found that negative JunD/AP-1 activity increases twofold during differentiation of SV40-transformed 3T3T cells (CSV3-1) and that decreasing JunD levels by antisense oligodeoxyribonucleotides significantly increases cell proliferation (31). These findings indicate that Jun/AP-1 transcription factors can be dissected into two parts: c-Jun/AP-1 and JunB/AP-1 positively regulate cell division, and JunD/AP-1 negatively regulates cell growth.
Our previous studies (16, 35, 36) have demonstrated that cell proliferation in the intestinal mucosa is dependent on the supply of polyamines to the dividing cells. Although the molecular mechanisms by which polyamines are required for the stimulation of cell proliferation are still unclear, changes in gene expression are known to occur (7, 17, 20). In this regard, we have recently reported that polyamine-deficient IEC-6 cells are associated with a significant decrease in expression of the protooncogenes c-fos, c-jun, and junB (36). On the basis of these results, we expected that polyamine depletion by treatment with DFMO would decrease AP-1 DNA binding activity in small intestinal crypt cells. The new finding described in this study, however, shows that inhibition of polyamine biosynthesis markedly increases AP-1 binding activity in the DFMO-treated cells (Fig. 2). The increased AP-1 DNA binding activities following polyamine depletion are specific because there are no significant differences in other transcription factors such as AP-2 and SP1 binding activities between control cells and polyamine-deficient cells (Fig. 4).
To determine whether the increased AP-1 activities following polyamine depletion result from either c-Jun/AP-1 and JunB/AP-1 or JunD/AP-1, we characterized the composition of increased AP-1 complexes in the DFMO-treated cells by performing gel supershift assays using various specific antibodies. As can be seen in Figs. 5 and 6, increased AP-1 complexes in polyamine-deficient cells are dramatically supershifted by the anti-JunD antibody but not by antibodies against c-Jun, JunB, or Fos proteins, suggesting that most of the increased AP-1 activities in the DFMO-treated cells are primarily contributed by an increase in JunD/AP-1. These findings are further supported by the observations that increased JunD/AP-1 binding activity in the DFMO-treated cells is paralleled by increases in the levels of both JunD mRNA and protein (Figs. 7 and 8). Increased levels of JunD protein following polyamine depletion are present just inside the nuclei of intestinal crypt cells (Fig. 9). These results clearly indicate that polyamine depletion significantly increases JunD/AP-1 binding activity and strongly support the hypothesis that intracellular polyamines are required for intestinal mucosal growth in association with their ability to modulate the balance between positive and negative Jun/AP-1 activities.
Because the importance of AP-1 transcription factors in the control of cell proliferation has been documented by numerous studies (2, 10) and because intracellular polyamines are essential for normal intestinal mucosal growth under physiological conditions (16, 19), the results presented in this paper raise a series of interesting questions for future study. The first question that should be addressed is the nature of the molecular mechanisms that activate the expression of the junD gene following polyamine depletion. Data presented in Fig. 7 show that the increased JunD protein level leading to increased JunD/AP-1 activity is paralleled by a significant increase in junD mRNA levels in IEC-6 cells exposed to DFMO for 4 and 6 days. It is not clear at present whether increased mRNA level for junD is due to an increase in gene transcription or results from the alteration of mRNA stabilization.
The second question is whether activated negative JunD/AP-1 plays a role in the growth inhibition process following polyamine depletion in intestinal crypt cells. Figure 10 clearly shows that increased JunD/AP-1 activity in DFMO-treated cells is accompanied by a significant decrease in cell growth that can be completely restored to normal by concomitant treatment with spermidine. Because JunD has a function opposed to c-Jun and JunB and promotes quiescence during the cell cycle in other cell types, the temporal association of increased expression of the junD gene and decreased cell division after polyamine depletion suggests that activated negative JunD/AP-1 activity and growth inhibition are causally related. In support of this possibility, JunD/AP-1 binding activity is not increased by exposure to a positive treatment of 5% dialyzed serum (Fig. 11). Our previous studies (36) have also demonstrated that inhibition of polyamine synthesis by DFMO significantly decreases steady-state levels of c-fos, c-jun, and junB mRNAs in IEC-6 cells. The repression of c-fos, c-jun, and junB expression in the DFMO-treated cells would be expected to facilitate the inhibitory effect of JunD on cell division because decreasing c-Jun/AP-1 and JunB/AP-1 activities would allow JunD/AP-1 activity to be dominant. Further studies are needed to determine whether decreasing JunD levels by using antisense RNA or by transfection of dominant negative JunD mutant will promote cell proliferation in polyamine-deficient cells.
The third issue raised by the current study is the interrelationship between c-Jun/AP-1, JunB/AP-1, and JunD/AP-1 activities during growth inhibition following polyamine depletion. Although activated JunD/AP-1 could function to prevent cells from entering the cell cycle in polyamine-deficient cells, the exact mechanism concerning how JunD/AP-1 functions in this regard is unclear. Possibilities include that negative JunD/AP-1 may compete with positive c-Jun/AP-1 or JunB/AP-1 for DNA binding or that JunD/AP-1 may regulate genes that are primarily involved in negatively controlling cell proliferation, whereas c-Jun/AP-1 and JunB/AP-1 may regulate genes that facilitate proliferation. Our previous studies (34, 36) and others (6) have found that expression of the c-fos gene decreases following polyamine depletion, suggesting a limited possibility that Jun proteins may dimerize with Fos. Because JunD is more abundant than c-Jun and JunB in the polyamine-deficient cells (Figs. 6 and 7), it may dimerize with c-Jun or JunB to suppress their stimulatory effects on cell proliferation.
In summary, these results indicate that polyamines are implicated in the regulation of Jun/AP-1 activities in intestinal epithelial cells. Depletion of intracellular polyamines by DFMO is associated with a significant increase in JunD/AP-1 activity, which is paralleled by increases in the levels of both JunD mRNA and protein in IEC-6 cells. Cell proliferation in the DFMO-treated cells is also significantly decreased. Because JunD/AP-1 has been shown to negatively regulate cell division in a variety of cell types, these findings suggest that polyamine depletion results in growth inhibition in the intestinal mucosa at least partially by altering expression of the junD gene and Jun/AP-1 binding activity.
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ACKNOWLEDGEMENTS |
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The authors thank Drs. Barbara L. Bass and Chin-Ming Wei for helpful discussion and Dr. Diane C. Fadely for critical reading of the manuscript.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-45314 and a Merit Review Grant from the Department of Veterans Affairs to J.-Y. Wang.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: J.-Y. Wang, Dept. of Surgery, Baltimore VA Medical Center and Univ. of Maryland, 10 North Greene St., Baltimore, MD 21201.
Received 31 March 1998; accepted in final form 21 October 1998.
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REFERENCES |
---|
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---|
1.
Abel, T.,
and
T. Maniatis.
Action of leucine zippers.
Nature
341:
24-25,
1989[Medline].
2.
Angel, P.,
and
M. Karin.
The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.
Biochim. Biophys. Acta
1072:
129-157,
1991[Medline].
3.
Bossy-Wetzel, E.,
R. Bravo,
and
D. Hanahan.
Transcription factors junB and c-jun are selectively up-regulated and functionally implicated in fibrosarcoma development.
Genes Dev.
6:
2340-2351,
1992[Abstract].
4.
Boyle, W. J.,
T. Smeal,
L. H. K. Defize,
P. Angel,
J. R. Woodgett,
M. Karin,
and
T. Hunter.
Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity.
Cell
64:
773-584,
1991.
5.
Bradford, M. A.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dying binding.
Anal. Biochem.
72:
224-254,
1976.
6.
Celano, P.,
S. B. Baylin,
and
R. A. Caserro.
Polyamines differentially modulate the transcription of growth-associated genes in human colon carcinoma cells.
J. Biol. Chem.
264:
8922-8927,
1989
7.
Celano, P.,
S. B. Baylin,
F. M. Diardiello,
B. D. Nelkin,
and
R. A. Casero.
Effect of polyamine depletion on c-myc expression in human colon carcinoma cells.
J. Biol. Chem.
263:
5491-5494,
1988
8.
Chirgwin, J. M.,
A. E. Przybyla,
R. J. MacDonald,
and
W. J. Rutter.
Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.
Biochemistry
18:
5294-5299,
1979[Medline].
9.
Harter, J. L.
Critical values for Duncan's new multiple range test.
Biometrics
16:
671-685,
1960.
10.
Herschman, H. R.
Primary response genes induced by growth factors and tumor promoters.
Annu. Rev. Biochem.
60:
281-319,
1991[Medline].
11.
Hirai, S. I.,
R. P. Ryseck,
F. Mechta,
R. Bravo,
and
M. Yaniv.
Characterization of junD: a new member of the jun proto-oncogene family.
EMBO J.
8:
1433-1439,
1989[Abstract].
12.
Janne, J.,
L. Alhonen,
and
P. Leinonen.
Polyamines: from molecular biology to clinical applications.
Ann. Med.
23:
241-259,
1991[Medline].
13.
Laemmli, U. K.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1990.
14.
Lasky, S. R.,
K. Iwata,
A. G. Rosmarin,
D. G. Caprio,
and
A. L. Maize.
Differential regulation of JunD by dihydroxycholecalciferol in human chronic myelogenous leukemia cells.
J. Biol. Chem.
270:
19676-19679,
1995
15.
Luk, G. D.,
L. J. Marton,
and
S. B. Baylin.
Ornithine decarboxylase is important in intestinal mucosal maturation and recovery from injury in rats.
Science
210:
195-198,
1980[Medline].
16.
McCormack, S. A.,
and
L. R. Johnson.
Role of polyamines in gastrointestinal mucosal growth.
Am. J. Physiol.
260 (Gastrointest. Liver Physiol. 23):
G795-G806,
1991
17.
Panagiotidis, C. A.,
S. Artandi,
K. Calame,
and
S. J. Silverstein.
Polyamines alter sequence-specific DNA-protein interactions.
Nucleic Acids Res.
23:
1800-1809,
1995[Abstract].
18.
Patel, A. R.,
J. Li,
B. L. Bass,
and
J.-Y. Wang.
Polyamine depletion is associated with an increase in negative jun/AP-1 activities in small intestinal crypt cells.
Gastroenterology
112:
A897,
1997.
19.
Patel, A. R.,
J. Li,
B. L. Bass,
and
J.-Y. Wang.
Expression of the transforming growth factor- gene during growth inhibition following polyamine depletion.
Am. J. Physiol.
275 (Cell Physiol. 44):
C590-C598,
1998
20.
Patel, A. R.,
and
J.-Y. Wang.
Polyamines modulate transcription but not posttranscription of c-myc and c-jun in IEC-6 cells.
Am. J. Physiol.
273 (Cell Physiol. 42):
C1020-C1029,
1997
21.
Pegg, A. E.
Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy.
Cancer Res.
48:
759-774,
1988[Abstract].
22.
Pfarr, C. M.,
F. Mechta,
G. Spyrou,
D. Lallemand,
S. Carillo,
and
M. Yaniv.
Mouse JunD negatively regulates fibroblast growth and antagonizes transformation by ras.
Cell
76:
747-760,
1994[Medline].
23.
Quaroni, A.,
J. Wands,
R. L. Trelstad,
and
K. J. Isselbacher.
Epithelial cell cultures from rat small intestine.
J. Cell Biol.
80:
248-265,
1979[Abstract].
24.
Rezzonico, R.,
A. Loubat,
D. Lallemand,
C. M. Pfarr,
D. F. Far,
A. Proudfoot,
B. Rossi,
and
G. Ponzio.
Cyclic AMP stimulates a JunD/Fra-2 AP-1 complex and inhibits the proliferation of interleukin-6-dependent cell lines.
Oncogene
11:
1069-1078,
1995[Medline].
25.
Ryder, K.,
A. Lanahan,
E. Perez-Albuerne,
and
D. Nathans.
junD: a third menber of the jun gene family.
Proc. Natl. Acad. Sci. USA
86:
1500-1503,
1989[Abstract].
26.
Ryder, K.,
L. F. Lau,
and
D. Nathans.
A gene activated by growth factors is related to the oncogene v-jun.
Proc. Natl. Acad. Sci. USA
85:
1487-1491,
1988[Abstract].
27.
Ryseck, R. P.,
S. I. Hirai,
and
R. Bravo.
Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts.
Nature
334:
535-537,
1988[Medline].
28.
Smith, M. J.,
and
E. V. Prochownik.
Inhibition of c-jun causes reversible proliferative arrest and withdrawal from the cell cycle.
Blood
79:
2107-2115,
1992[Abstract].
29.
Tourita, Y.,
N. Toshikazu,
and
A. Ichihara.
Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes.
Exp. Cell Res.
135:
363-371,
1981[Medline].
30.
Wang, H.,
and
R. E. Scott.
Adipocyte differentiation selectively represses the serum inducibility of c-jun and junB by reversible transcription-dependent mechanisms.
Proc. Natl. Acad. Sci. USA
91:
4649-4653,
1994[Abstract].
31.
Wang, H.,
Z. Xie,
and
R. E. Scott.
Differentiation modulates the balance of positive and negative Jun/AP-1 activities to regulate cellular proliferative potential: different effects in nontransformed and transformed cells.
J. Cell Biol.
135:
1151-1162,
1996[Abstract].
32.
Wang, J.-Y.,
and
L. R. Johnson.
Luminal polyamines stimulate repair of gastric mucosal stress ulcers.
Am. J. Physiol.
259 (Gastrointest. Liver Physiol. 22):
G584-G592,
1990
33.
Wang, J.-Y.,
and
L. R. Johnson.
Polyamines and ornithine decarboxylase during repair of duodenal mucosa after stress damage in rats.
Gastroenterology
100:
333-343,
1991[Medline].
34.
Wang, J.-Y.,
and
L. R. Johnson.
Expression of protooncogenes c-fos and c-myc in healing of gastric mucosal stress ulcers.
Am. J. Physiol.
266 (Gastrointest. Liver Physiol. 29):
G878-G886,
1994
35.
Wang, J.-Y.,
S. A. McCormack,
M. J. Viar,
and
L. R. Johnson.
Stimulation of proximal small intestinal mucosal growth by luminal polyamines.
Am. J. Physiol.
261 (Gastrointest. Liver Physiol. 24):
G504-G511,
1991
36.
Wang, J.-Y.,
S. A. McCormack,
M. J. Viar,
H. Wang,
C.-C. Tzen,
R. E. Scott,
and
L. R. Johnson.
Decreased expression of protooncogenes c-fos, c-myc, and c-jun following polyamine depletion in IEC-6 cells.
Am. J. Physiol.
265 (Gastrointest. Liver Physiol. 28):
G331-G338,
1993
37.
Wei, C.-M.,
A. L. Clavell,
and
J. C. Burnett.
Atrial and pulmonary endothelin mRNA is increased in a canine model of chronic low cardiac output.
Am. J. Physiol.
273 (Regulatory Integrative Comp. Physiol. 42):
R838-R844,
1997
38.
Ye, Z.-S.,
and
H. H. Samuels.
Cell- and sequence-specific binding of nuclear proteins to 5'-flanking DNA of the rat growth hormone gene.
J. Biol. Chem.
262:
6313-6317,
1987