Departments of 1 Physiology and Biophysics and 2 Pharmacology, University of Tennessee, Memphis, College of Medicine, Memphis, Tennessee 38163
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Our laboratory has shown that asparagine (ASN)
stimulates both ornithine decarboxylase (ODC) activity and gene
expression in an intestinal epithelial cell line (IEC-6). The effect of
ASN is specific, and other A- and N-system amino acids are almost as
effective as ASN when added alone. In the present study, epidermal growth factor (EGF) was unable to increase ODC activity in cells maintained in a salt-glucose solution (Earle's balanced salt
solution). However, the addition of ASN (10 mM) in the presence of EGF
(30 ng/ml) increased the activity of ODC 0.5- to 4-fold over that stimulated by ASN alone. EGF also showed induction of ODC with glutamine and -aminoisobutyric acid, but ODC induction was maximum with ASN and EGF. Thus the mechanism of the interaction between ASN and
EGF is important for understanding the regulation of ODC under
physiological conditions. Therefore, we examined the expression of the
ODC gene and those for
several protooncogenes under the same conditions. Increased expression
of the genes for c-Jun and c-Fos but not for ODC occurred
with EGF alone. The addition of ASN did not further increase the
expression of the protooncogenes, but the combination of EGF and ASN
further increased the expression of ODC over that of ASN alone. Western
analysis showed no significant difference in the level of ODC protein
in Earle's balanced salt solution, ASN, EGF, or EGF plus ASN. Addition
of cycloheximide during ASN and ASN plus EGF treatment completely
inhibited ODC activity without affecting the level of ODC protein.
These results indicated that 1) the
increased expression of protooncogenes in response to EGF is
independent of increases in ODC activity and 2) potentiation between EGF and ASN
on ODC activity may not be due to increased gene transcription but to
posttranslational regulation and the requirement of ongoing protein
synthesis involving a specific factor dependent on ASN.
epidermal growth factor; polyamines; amino acids; c-Fos; c-Jun; enzyme activity
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE POLYAMINES SPERMIDINE and spermine and their precursor putrescine are required for cell growth and proliferation of eukaryotic cells (7, 40, 47). The intracellular levels of polyamines are highly regulated and primarily depend on the activity of ornithine decarboxylase (ODC; EC 4.1.17), which catalyzes the first rate-limiting step in polyamine synthesis (25-27). ODC activity is regulated differently in different tissues and cells, but, in general, activity increases dramatically and transiently in response to trophic agents, other proliferative stimuli, and damage (7, 11, 13, 18, 27). For these reasons, there is considerable interest in the regulation of the activity of ODC, and the enzyme has been investigated at the level of gene transcription, mRNA translation, protein turnover, and inhibitors of enzyme activity (2, 12, 14, 18, 36).
For a number of years, our laboratory has been interested in the role
of polyamines in the growth and repair of the gastrointestinal mucosa
and has examined these processes in both rats and cultured, normal
intestinal epithelial cells. The mammalian gastrointestinal epithelium
is especially suited to study the control of proliferation because of
its rapid and continuous renewal. Inhibition of ODC by
-difluoromethylornithine depletes polyamines and inhibits the growth
of cultured IEC-6 cells, a nontransformed line originally developed
from fetal rat crypt cells (29), and prevents the normal repair of
mucosal stress ulcers (45, 46, 49). The addition of exogenous
polyamines in both model systems restores cell growth and healing to
normal levels.
Basal levels of ODC are quite low, but enzyme activity increases rapidly and dramatically in response to the appropriate stimuli. The enzyme has one of the shortest half-lives of any mammalian enzyme (2, 3). Stimulation of ODC activity in small intestinal mucosa by luminal contents has been well documented. Among normal dietary constitutents, some amino acids have been shown to markedly increase ODC activity and mucosal growth when administered intragastrically (22, 24). In 1971, Fausto (7) showed that some amino acids could increase ODC activity in regenerating liver. Chen and Canellakis (3) and Chen and Chen (4) examined the ability of amino acids to induce ODC activity in cells incubated in a simple salt-glucose solution, thus eliminating stimulation from growth factors present in serum or other amino acids and nutrients normally found in incubation medium. They showed that ODC activity was increased in neuroblastoma cells by asparagine (ASN) and glutamine. Additional studies by the same group (32, 33, 42) showed that, although ASN was the most effective, other amino acids transported by one of the Na+-dependent systems could also induce ODC activity. Induction of ODC activity was shown to be dependent on Na+ and independent of metabolism or the ability of the amino acid to be incorporated into protein. Of particular interest was the finding, in a variety of cell types, that the induction of ODC by hormones and growth factors required the presence of ASN or a similar amino acid (32, 33). Neither epidermal growth factor (EGF), nerve growth factor, platelet-derived growth factor, nor insulin was able to induce ODC activity in a salt-glucose solution unless ASN was present in the medium. Wang et al. (48) have shown previously that ASN increases ODC activity and DNA synthesis in IEC-6 cells in growth medium. The increase was accompanied by an increase in ODC mRNA, which was due partly to a decrease in the rate of degradation. EGF also stimulated ODC activity in these cells as well as increased growth and levels of several protooncogenes. Expression of some protooncogenes in IEC-6 cells is inhibited by polyamine depletion (47).
The primary purpose of these studies was to examine the interaction between EGF and ASN in the regulation of ODC activity in IEC-6 cells. We demonstrate that EGF does not increase ODC activity when given in a salt-glucose solution, whereas ASN is able to do so. Furthermore, EGF does potentiate the effects of ASN. We also examined this process of potentiation to discern at which level it occurs. As the studies progressed, it became obvious that we could use the salt-glucose model as a tool to examine whether the effects of EGF required the induction of ODC activity. Thus we show that EGF increases the expression of c-fos and c-jun mRNA in a salt-glucose solution, indicating that this stimulation does not require increases in ODC activity. Two different mechanisms of activation of ODC are indicated because ASN and EGF show potentiation, and this was proven with rapamycin, which blocks serum- and EGF-induced enzyme activity but does not alter the effect of ASN.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chemicals and supplies. Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (FBS) were from GIBCO BRL (Grand Island, NY). Biochemicals were purchased from Sigma (St. Louis, MO). L-[1-14C]ornithine (specific activity 51.6 mCi/mmol) was purchased from DuPont NEN (Boston, MA). The primary antibody was an affinity-purified rabbit polyclonal antibody against ODC and was a kind gift from Dr. M. K. Haddox (University of Texas at Houston) (9). Mouse monoclonal antibody against ODC was purchased from Sigma. Western blot detection systems were purchased from DuPont NEN.
Cell culture. The IEC-6 cell line was obtained from the American Type Culture Collection (Manassas, VA) at passage 13. The stock was maintained in T-150 flasks in a humidified, 37°C incubator in an atmosphere of 90% air-10% CO2. The medium consisted of Dulbecco's modified Eagle's medium (DMEM; GIBCO BRL) with 5% heat-inactivated FBS, 10 µg insulin, and 50 µg gentamicin sulfate/ml. The stock was passaged weekly at 1:20 and fed three times per week. Passages 15-20 were used in the experiments.
Experimental protocol. For most experiments, the cells were taken up with 0.05% trypsin plus 0.53 mM EDTA · 4Na in Hanks' balanced salt solution without calcium and magnesium. They were counted in a hemocytometer and plated (day 0) at 6.25 × 104 cells/cm2 in DMEM plus 5% dialyzed FBS, 10 µg insulin, and 50 µg gentamicin sulfate/ml. The cells were fed on day 2. On day 3, the medium was removed, the cells were washed once with Hanks' balanced salt solution, and the medium was replaced with serum-free DMEM with 10 µg insulin and 50 µg gentamicin sulfate/ml. On day 4, the medium was removed, the cells were washed once with Hanks' balanced salt solution, and the medium was replaced with Earle's balanced salt solution (EBSS) containing whatever additions were required by the experiment. For experiments involving cell harvesting for Northern blot analysis, the protocol was modified slightly as follows. The serum starvation on day 3 was done in EBSS rather than in DMEM, and, on day 4, treatments were added as concentrated solutions directly into EBSS without changing the medium.
ODC assay. The activity of the enzyme
ODC was assayed with a radiometric technique in which the amount of
14CO2
liberated from
DL-[1-14C]ornithine
was estimated (31). Briefly, after experimental treatment, the dishes
were placed on ice; the monolayers were washed three times with cold
Dulbecco's PBS (DPBS); and 0.5 ml of 1 mM Tris buffer (pH 7.4)
containing 1 mM EDTA, 0.05 mM pyridoxal phosphate, and 5 mM
dithiothreitol was added. The cells were frozen at 80°C
until assayed. At the time of assay, the cells were thawed on ice,
scraped into Microfuge tubes, sonicated, and centrifuged at 12,000 g at 4°C for 10 min. The ODC
activity of an aliquot of supernatant was incubated in a stoppered tube
in the presence of 6.8 nmol of
[14C]ornithine for 15 min at 37°C. The
14CO2
liberated by the decarboxylation of ornithine was trapped on a piece of
filter paper impregnated with 20 µl of 2 N NaOH, which was suspended
in a center well above the reaction mixture. The reaction was stopped
by the addition of TCA to a final concentration of 5%. The
14CO2
trapped in the filter paper was measured by liquid scintillation spectroscopy. Aliquots of the supernatant were assayed for total protein with the method described by Bradford (1). Enzymatic activity
is expressed as picomoles of CO2
per hour per milligram of protein.
RNA isolation and Northern blot
analysis. Total RNA was extracted with a guanidium
isothiocyanate solution and purified by CsCl density gradient
ultracentrifugation as described by Chirgwin et al. (6). Briefly, the
monolayer of cells was washed with DPBS and lysed in 4 M guanidium
isothiocyanate. The lysates were centrifuged through a 5.7 M CsCl
cushion at 150,000 g at 18°C for
20 h. After centrifugation, the supernatant was aspirated, and the
resulting RNA pellet was dissolved in 10 mM
Tris · HCl (pH 7.4) containing 5 mM EDTA and 1%
sodium dodecyl sulfate (SDS). 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. Final RNA was dissolved in water and
estimated from its ultraviolet absorbance at 260 mm, with a conversion
factor of 40 optical density units. Total RNA (30 µg) was denatured
and fractionated electrophoretically with a 1.2% agarose gel
containing 3% formaldehyde and transferred by blotting to a
nitrocellulose membrane. The blot was prehybridized for 24 h at
42°C with 5× Denhardt's solution, 5× saline-sodium
citrate (SSC), 50% formamide, 25 mM potassium phosphate, and 50 µg/ml of denatured salmon sperm DNA. Hybridization was carried out
overnight at 4°C in the same solution containing 10% dextran
sulfate and DNA probes, which were labeled with
[-32P]dCTP with a
standard nick translation procedure. Blots were washed once with
1× SSC-0.1% SDS at room temperature for 10 min, followed by two
changes of 0.25× SSC-0.1% SDS, the first at 42°C for 25 min
and the second at room temperature for 10 min. After the final wash,
the membrane was autoradiographed with intensifying screens at
80°C.
Western blot analysis of ODC protein. After experimental treatments, IEC-6 cells were washed twice with DPBS. The DPBS was removed, 500 µl of radioimmunoprecipitation assay buffer (50 mM Tris · HCl, pH 7.4, 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 mM NaF, and 1 µg/ml each of aprotinin, leupeptin, and pepstatin) were added, and the plates were rotated at 4°C for 30 min and scraped. The cell lysate was centrifuged at 10,000 rpm for 10 min, and the clear supernatant was used to determine the protein concentration by the method of Bradford (1). Total cell protein (500 µg) was immunoprecipitated with anti-mouse ODC antibody separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane for Western blotting with anti-rabbit ODC antibody. Immune complexes were visualized with a chemiluminescence detection system (DuPont NEN).
[3H]leucine incorporation. Cells were grown in DMEM as described in Experimental protocol. The cells were then incubated in EBSS with ASN and/or EGF, 5 µCi/ml of [3H]leucine were added after 2.5 h, and the cells were further incubated for 30 min. The cells were washed twice with DPBS and lysed with radioimmunoprecipitation assay buffer as described in Western blot analysis of ODC protein. The amount of radioactivity in the cell extract was determined. Proteins equivalent to 100,000 counts/min from each sample were precipitated by 10% TCA and separated by 10% SDS-PAGE. Detection of radioactive proteins was carried out with the use of ENHANCE (DuPont NEN) reagent following the manufacturer's instructions.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Synergistic effect of ASN and EGF on ODC
activity. IEC-6 cells were cultured in DMEM as
described in MATERIALS AND METHODS. The medium was replaced with EBSS containing varying concentrations of
ASN with and without 30 ng/ml of EGF. Basal ODC activity was nearly
undetectable. ASN in doses ranging from 1 to 100 mM significantly induced ODC activity, with a peak effect occurring at a concentration of 10 mM (Fig. 1). EGF by
itself had no effect on enzyme activity but when given with ASN caused
a significant increase in ODC activity over and above that induced by
the comparable dose of ASN. As shown in Fig.
2, glutamine (N transport system) and
-aminoisobutyric acid (AIB; A type) also increased ODC activity,
which was increased further by EGF. The potentiation by EGF, however,
was greater with ASN than with these other two amino acids. Amino acids
transported by other systems were ineffective because valine (L type)
and lysine (Y+ type) caused no
increase in ODC activity.
|
|
Effects on ODC mRNA and protein
levels. To gain some insight into the synergy between
ASN and EGF in their regulation of ODC activity, we examined ODC mRNA
and protein levels under the same set of experimental conditions.
Figure 3 shows that ASN increased ODC mRNA
more than threefold, whereas EGF had little effect by itself. When EGF
was added to EBSS containing ASN, however, mRNA levels increased more
than fivefold. Even though EGF had no effect on ODC activity or ODC
mRNA levels, it increased c-fos and
c-jun mRNA. ASN had no effect on the
mRNA levels of these two protooncogenes, and there was no further
increase in mRNA when ASN and EGF were combined.
|
Western analysis of ODC protein showed little correlation with the
observed changes in either enzyme activity or ODC mRNA (Fig.
4). IEC-6 cell extracts contained a protein
band corresponding to the 53-kDa molecular mass of a
monomer of ODC (Fig. 4A). The identity of this band was confirmed by loading preimmune serum onto the
gel, which showed no band corresponding to ODC, and by immunoprecipitation with anti-mouse ODC antibody (Fig.
4B). Results in Fig. 4 show no
difference in ODC protein in any of the treatment groups compared with
the control group or with each other. These data were further supported
by the incorporation of
[3H]leucine into the
cytoplasmic fraction of IEC-6 cells under the same experimental
conditions. Although EGF increased the overall rate of protein
synthesis, none of the treatment groups showed an apparent change in
the intensity of any particular band and specifically in the 53-kDa
region (Fig. 5). Immunoprecipitation of
ODC from the labeled extracts failed to detect ODC
protein on the same gel.
|
|
Stability of ODC activity, mRNA, and
protein. As a possible explanation for the combined
effects of ASN and EGF, we next examined whether EGF altered the
stability of ODC activity, mRNA, and protein in the presence of ASN. As
shown in Fig. 6, the addition of EGF to
EBSS containing ASN increased the half-life of ODC activity from 46 to
66 min. Because ODC activity is undetectable in EBSS alone or in EBSS
to which EGF has been added, we were only able to compare these two
groups. Examination of the half-life of ODC mRNA under the same
conditions showed no difference between ASN alone and ASN plus EGF
(Fig. 7).
|
|
Although treatment with cycloheximide for 2 h resulted in 84 and 76%
losses in ODC activity in cells stimulated with ASN and ASN plus EGF,
respectively, Western blot analysis showed no difference in the levels
of ODC protein in the same experiment (Fig. 8). In the
absence of cycloheximide, cells stimulated by ASN alone lost 45% of
their ODC activity in 2 h, whereas those stimulated with both ASN and
EGF lost only 20% of their activity. Figure 8 also shows the
disappearance of the ODC protein band from preimmune serum.
|
Effect of cycloheximide and actinomycin D on the
induction of ODC activity. Even though changes in mRNA
levels correlated fairly well with the induction of ODC activity after
ASN, EGF, and the combination of the two, it is obvious from Fig. 4
that these were not reflected by the amount of ODC protein present. In
all cases, including unstimulated control cells, the levels of enzyme
protein were approximately the same. These data suggest the involvement
of posttranslational processes in the induction of ODC activity. To
determine the involvement of protein synthesis in the induction of
enzyme activity, cells were exposed to cycloheximide at the same time
that they were stimulated by either ASN, that EGF, or the two agents
combined. Table 1 shows that cycloheximide prevented any
enzyme stimulation by ASN and by the combination of ASN and EGF. Thus
the synthesis of new protein is required for the induction of ODC
activity. Table 1 also shows that enzyme activation is prevented by
actinomycin D, indicating that transcription is also required for the
stimulation of ODC activity.
|
Effect of rapamycin on the induction of ODC
activity. To distinguish the mechanisms of the
induction of ODC by serum and ASN in a salt-glucose solution, basal ODC
activity was induced by 10 mM ASN and 10% FBS in the presence and
absence of 100 nM rapamycin, a selective repressor of translation
initiation of an mRNA with a polypyrimidine-rich tract that includes
ODC mRNA. Figure 9 shows that 10 mM ASN and 10% FBS
increased ODC activity >800- and 200-fold, respectively, when added
alone for 3 h. The addition of rapamycin along with 10% FBS
significantly repressed the ODC induction by 80%; however,
ASN-mediated induction was not affected by rapamycin. This result
clearly indicates that the mechanism of ODC induction in salt-glucose
solution is a distinct phenomenon and further supports the
posttranslational control of ODC enzyme activity induction by ASN.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A variety of evidence indicates that at least two different mechanisms are involved in the stimulation of ODC activity. Several stimuli increase ODC mRNA in both tissues and cultured cells, but, in many cases, the increase in mRNA does not account for the magnitude of induction of ODC activity (44). Ginty et al. (8) found that increased levels of active ODC protein and enzyme activity were sensitive to putrescine and to a calmodulin antagonist, whereas other processes regulating ODC mRNA were insensitive to these compounds. The findings that ASN but not growth factors can increase ODC activity in a salt-glucose solution and that growth factors will, nevertheless, potentiate the induction of enzyme activity by ASN also argue strongly for independent signaling pathways for the regulation of ODC.
We conducted all of the current experiments in EBSS with glucose added to separate the effects of ASN from those of growth factors. In previous experiments designed to investigate the mechanism of regulation of ODC activity, investigators added growth factors or serum to cells in culture medium that, of course, contained a full complement of amino acids. As a result, all mechanisms were activated simultaneously.
In our present experiments, ASN caused a dose-dependent increase in ODC activity and EGF was without effect when given by itself. However, EGF significantly increased the stimulation provided by ASN (Fig. 1). Although other amino acids transported by A- and N-type systems increased ODC activity and showed synergy with EGF, ASN was the most effective (Fig. 2). All of these results (Figs. 1 and 2) agree with previous reports in other cell types (4, 5, 15, 19).
A nonmetabolizable amino acid analog, AIB, also stimulated ODC activity, indicating that the effects of amino acids are direct and not caused by subsequent processing. The response to AIB was also increased further by the addition of EGF (Fig. 2). These data indicate that the synergism with EGF is specific for A- and/or N-type amino acids and suggests that ASN or a similar amino acid must be present for the maximal induction of ODC activity. Kandil et al. (17) have also shown that glutamine and ASN stimulated ODC activity and proliferation but that AIB was ineffective in a porcine jejunal enterocyte cell line. Chen and Chen (5) found a greater induction of ODC activity in ras-transformed 3T3 cells compared with their nontransformed counterparts, suggesting the involvement of an additional cellular factor for maximal induction.
The effects of ASN and EGF on the level of ODC mRNA in EBSS were qualitatively similar to their effects on ODC activity. ASN by itself increased ODC mRNA ~3.5-fold, whereas EGF stimulated a nonsignificant 40% increase. In the presence of ASN, however, EGF further increased ODC mRNA to 5.5 times the basal level (Fig. 3). Upregulation of primary growth response genes, the protooncogenes c-fos and c-jun, which encode transcription factors, in response to EGF may result in activation of the ODC gene via activator protein-1 (23). Increased levels of ODC gene transcription and enzyme activity by c-myc have been demonstrated in BALB/c, 3TC, and rat 1a fibroblasts (43). In our study, however, EGF increased c-fos and c-jun mRNA expression even though it had no significant effect on ODC mRNA. Thus the stimulation of these protooncogenes by EGF does not depend on its ability to increase ODC activity. In contrast, although ASN significantly increased both ODC mRNA and activity, it had no effect on the expression of c-fos and c-jun. Taken together, our Northern analyses indicate a mutual independence between ODC induction and protooncogene transcription in IEC-6 cells.
The significant potentiation of ASN-induced ODC activity by EGF could be due to a variety of events, including posttranscriptional stabilization of ODC mRNA, recruitment of ODC mRNA from a nontranslatable pool, ODC enzyme synthesis (translation), or posttranslational modification (activation) of the enzyme. Regulation of ODC is cell or tissue specific and may involve one or more of the above mechanisms, depending on the polyamine requirements for function of the cells or tissue in question (16, 20). The stability of ODC mRNA in cells treated with both ASN and EGF appeared to be the same as in those treated with ASN alone (Fig. 7), ruling out posttranscriptional stabilization of ODC mRNA as an explanation for the synergistic induction of ODC activity.
Changes in translation have been suggested in several other systems for the regulation of ODC activity (39). ODC exists in an equilibrium between active dimers and inactive monomers (38, 41). In its active form, mammalian ODC is a homodimer, whereas the 53-kDa monomer retains no enzymatic activity. In a recent study, Pegg and Williams-Ashmann (28) presented biochemical evidence that the active site of mammalian ODC interfaces between the two subunits that form the active dimer. Moreover, Tobias and Kahana (41) showed that each subunit has a split active site, with the two residues participating in the active site being located on different segments. These observations established the relationship between structural properties and enzymatic activity and suggest that the level of the 53-kDa monomer does not indicate the level of active ODC protein in the cell extract but only the level of inactive ODC protein. The level of ODC protein measured as the 53-kDa protein did not change significantly in cells treated with ASN or in those treated with ASN plus EGF compared with EBSS or EGF alone (Fig. 4). This observation was further supported by [3H]leucine incorporation, which showed that stimulation of ODC activity in IEC-6 cells by ASN or ASN plus EGF was not due to the stimulation of general protein synthesis. EGF significantly increased [3H]leucine incorporation 52% compared with that in the control group (Fig. 5A). Immunoprecipitation of ODC from [3H]leucine-labeled cell extracts failed to show an ODC protein band corresponding to 53 kDa. A fluorogram of labeled protein showed no gross changes in the intensity of labeling of any protein band in the 53-kDa region (Fig. 5B). These results clearly indicate that the induction of ODC activity by ASN or ASN plus EGF does not involve new ODC protein synthesis.
Poor translatability of ODC mRNA in vitro has also been reported and
suggested to be due to an extensive secondary structure at the
5'-end of the ODC mRNA. The eukaryotic initiation factor eIF-4F
participates in the melting of mRNA secondary structures (10, 21).
Overexpression of eIF-4F increases the translation efficiency of mRNA
with a secondary structure, including ODC mRNA (37). It is possible
that the induction of ODC by ASN plus EGF may involve eIF-4F and
increased rates of translation of the
ODC gene. Seidel and Ginty
(34) have shown that the addition of FBS to the medium
induced ODC activity in IEC-6 cells and that this was accompanied by a
parallel increase in the level of ODC protein as determined by
[-3H]difluoromethylornithine
binding, which measures only active enzyme protein. The addition of
putrescine along with serum decreased ODC activity, with a concomitant
decrease in ODC protein content. Seidel and Ragan (35) further
established the role of eIF-4F in the induction of ODC activity in
IEC-6 cells by using rapamycin, a specific inhibitor of eIF-4F-mediated
translation repression. Surprisingly, only 50% of the serum-induced
ODC activity was inhibited by rapamycin, strongly suggesting that more
than one mechanism is involved in the induction of ODC activity and
that the mechanism depends on the type of stimulus. This observation
prompted us to investigate the effect of rapamycin on ASN-mediated ODC
induction. Figure 9 shows that rapamycin did not inhibit the induction
of ODC activity mediated by ASN, whereas serum-induced enzyme activity was inhibited 85% by the same dose. The failure of rapamycin to block
the response to serum entirely can be ascribed to the presence of amino
acids in the serum as well as to the growth factors, the effects of
which were inhibited. The reason Seidel and Ragan saw only 50%
inhibition in response to serum is probably because their studies were
conducted in medium that supplies a full complement of amino acids.
These results rule out the possibility of the involvement of eIF-4F and
new protein synthesis in the induction of ODC activity by ASN and
establish that ODC induction by ASN is a distinct process from that of
growth factors.
The half-life of ODC activity was 66 min in the presence of both ASN and EGF compared with 46 min with ASN alone (Fig. 6). A 76-84% decrease in induced ODC activity after a 2-h cycloheximide treatment was not accompanied by a decrease in ODC protein (Fig. 8). Thus, in the absence of ongoing protein synthesis, ODC activity induced by either ASN alone or the combination of ASN and EGF declines rapidly.
The decay in ODC activity was only 26% slower in the absence of cycloheximide in cells treated with ASN plus EGF compared with those treated with ASN alone, which was not significant. These observations indicate the involvement of an additional protein factor for the assembly or stabilization of the active conformation of the ODC protein. Protein turnover is low in IEC-6 cells, and ODC protein may be subject to posttranslational control, causing rapid activation-inactivation cycles in response to proliferative stimuli.
ODC exists in both phosphorylated and unphosphorylated forms in RAW 264 cells (30). Phosphorylated ODC was more stable and had a greater catalytic efficiency and higher maximal velocity, demonstrating the requirement of an as yet unidentified protein kinase in the induction of ODC activity. Chen and Canellakis (3) reported that, in mouse N18 neuroblastoma cells, the addition of ASN increased the half-life of ODC from 12.5 to 420 min. The addition of actinomycin D along with ASN had no effect, whereas the addition of cycloheximide with ASN inhibited the induction of ODC activity. In contrast, in our study, the induction of ODC activity was inhibited by both actinomycin D and cycloheximide (Table 1), indicating the necessity of both transcription and translation and suggesting the requirement of de novo protein synthesis.
In summary, we have shown the presence of two synergistic mechanisms for maximal induction of ODC activity. Stimulation by EGF involved eIF-4F, whereas activation of ODC by ASN did not. Although induction of ODC activity did not involve the synthesis of ODC protein, it did require both transcription and translation. These results indicate that a specific inducible factor transcribed and translated in the presence of ASN and EGF is essential for the induction of ODC activity. We suggest that this factor is part of the structural requirement for the formation of the active homodimer of ODC, indicating the mechanism for the posttranslational regulation of the enzyme.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. M. K. Haddox (University of Texas at Houston) for the ornithine decarboxylase antibody, Pat Blanner for technical assistance, and Danny Morse for help with the figures.
![]() |
FOOTNOTES |
---|
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-16505 (to L. R. Johnson) and National Heart, Lung, and Blood Institute Grant HL-48308 (to T. B. Patel).
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 and other correspondence: R. M. Ray, Dept. of Physiology and Biophysics, Univ. of Tennessee, Memphis, 894 Union Ave. Memphis, TN 38163 (Email: rray{at}physio1.utmem.edu).
Received 14 July 1998; accepted in final form 1 December 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bradford, M. A.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal. Biochem.
72:
224-254,
1976.
2.
Canellakis, E. S.,
D. Viceps-Madore,
D. A. Kariakidis,
and
J. S. Heller.
The regulation and function of ornithine decarboxylase and of polyamines.
Curr. Top. Cell. Regul.
15:
155-202,
1979[Medline].
3.
Chen, K. Y.,
and
E. S. Canellakis.
Enzyme regulation in neuroblastoma cells in a salt/glucose medium: induction of ornithine decarboxylase by asparagine and glutamine.
Proc. Natl. Acad. Sci. USA
74:
3791-3795,
1977[Abstract].
4.
Chen, Z. P.,
and
K. Y. Chen.
Mechanism of regulation of ornithine decarboxylase gene expression by asparagine in a variant mouse neuroblastoma cell line.
J. Biol. Chem.
267:
6946-6951,
1992
5.
Chen, Z. P.,
and
K. Y. Chen.
Asparagine markedly induces the expression of ornithine decarboxylase gene in transformed mammalian cells but not in their untransformed counterpart.
Cancer Lett.
86:
97-103,
1994[Medline].
6.
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].
7.
Fausto, N.
The control of ornithine decarboxylase activity during liver regeneration.
Biochim. Biophys. Acta
238:
116-128,
1971[Medline].
8.
Ginty, D. D.,
M. Marlowe,
P. H. Pekala,
and
E. Seidel.
Multiple pathways for the regulation of ornithine decarboxylase in intestinal epithelial cells.
Am. J. Physiol.
258 (Gastrointest. Liver Physiol. 21):
G454-G460,
1990
9.
Greenfield, A. R. L.,
S. M. Taffet,
and
M. K. Haddox.
Immunocytochemical localization of ornithine decarboxylase in cultured murine cells.
Cell Tissue Res.
243:
33-40,
1986[Medline].
10.
Grens, A.,
and
I. E. Scheffler.
The 5'- and 3'-untranslated region of ornithine decarboxyase mRNA affect the translational efficiency.
J. Biol. Chem.
265:
11812-11816,
1990.
11.
Hayashi, S.
Ornithine decarboxylase.
In: Biology, Enzymology, and Molecular Genetics, edited by S. Hayashi. Elmsford, NY: Pergamon, 1989, p. 35-46.
12.
Holtta, E.,
and
P. Pohjanpelto.
Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine and spermine.
J. Biol. Chem.
261:
9502-9508,
1986
13.
Hovis, J. G.,
D. J. Stumpo,
D. L. Halsey,
and
P. J. Blackshear.
Effect of mitogen on ornithine decarboxylase activity and messenger RNA level in normal and protein kinase-C deficient NIH-3T3 fibroblasts.
J. Biol. Chem.
261:
10380-10386,
1986
14.
Kahana, C.,
and
D. Nathan.
Translational regulation of mammalian ornithine decarboxylase by polyamines.
J. Biol. Chem.
260:
15390-15393,
1985
15.
Kanamoto, R.,
S. M. Boyle,
T. Oka,
and
S. Hayashi.
Molecular mechanism of the synergistic induction of ornithine decarboxylase by asparagine and glucagon in primary cultured hepatocytes.
J. Biol. Chem.
262:
14801-14805,
1987
16.
Kanamoto, R.,
M. Nishiyama,
S. Matsufuji,
and
S. Hayashi.
Translational control mechanism of ornithine decarboxylase by asparagine and putrescine in primary cultured hepatocytes.
Arch. Biochem. Biophys.
291:
247-254,
1991[Medline].
17.
Kandil, H.,
R. Argenzio,
W. Chen,
H. Berschneider,
A. Stiles,
J. Westwick,
R. Rippe,
D. Brenner,
and
J. Rhoads.
L-Glutamine and L-asparagine stimulate ODC activity and proliferation in a porcine jejunal enterocyte line.
Am. J. Physiol.
269 (Gastrointest. Liver Physiol. 32):
G591-G599,
1995
18.
Kontula, K. K.,
T. K. Torkkeli,
C. W. Bardin,
and
O. A. Janne.
Androgen induction of ornithine decarboxylase mRNA in mouse kidney as studied by complementary DNA.
Proc. Natl. Acad. Sci. USA
81:
731-735,
1984[Abstract].
19.
Law, C. L.,
and
W. F. Fong.
Asparagine transport and the induction of ornithine decarboxylase in H-35 rat hepatoma cells.
Biochem. Int.
14:
667-674,
1987[Medline].
20.
Lovkvist, E.,
L. Stjernborg,
and
L. Persson.
Feedback regulation of mammalian ornithine decarboxylase. Studies using transient expression system.
Eur. J. Biochem.
215:
753-759,
1993[Abstract].
21.
Manzella, J. M.,
and
P. J. Blackshear.
Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region.
J. Biol. Chem.
265:
11817-11822,
1990
22.
Maudsley, D. V.,
J. Leif,
and
Y. Kobayashi.
Ornithine decarboxylase in rat small intestine: stimulation with food or insulin.
Am. J. Physiol.
231:
1557-1561,
1976[Medline].
23.
McConlogue, L.,
G. Gupta,
L. Wu,
and
P. Coffino.
Molecular cloning and expression of the mouse ornithine decarboxylase gene.
Proc. Natl. Acad. Sci. USA
81:
540-544,
1984[Abstract].
24.
Minami, H.,
K. Miyamoto,
Y. Fuji,
Y. Nakabou,
and
H. Higara.
Induction of intestinal ornithine decarboxylase by single amino acid feeding.
J. Biochem. (Tokyo)
98:
133-139,
1985[Abstract].
25.
Pegg, A. E.
Polyamine metabolism and its importance in neoplastic growth and as a target for chemotherapy.
Cancer Res.
48:
759-774,
1986[Abstract].
26.
Pegg, A. E.
Recent advances in the biochemistry of polyamines in eukaryotes.
Biochem. J.
234:
249-262,
1986[Medline].
27.
Pegg, A. E.,
and
P. P. McCann.
Polyamine metabolism and function.
Am. J. Physiol.
243 (Cell Physiol. 12):
C212-C221,
1982[Abstract].
28.
Pegg, A. E.,
and
H. G. Williams-Ashmann.
Biosynthesis of putrescine.
In: Polyamines in Biology and Medicine, edited by D. R. Morris,
and L. J. Morton. New York: Dekker, 1981, p. 4-42.
29.
Quaroni, A.,
J. Wands,
R. L. Trelstad,
and
K. J. Isselbacher.
Epithelial cell culture from rat small intestine.
J. Cell Biol.
80:
248-265,
1988[Abstract].
30.
Reddy, S. G.,
S. M. McIlheran,
B. J. Cochran,
L. L. Worth,
L. A. Bishop,
P. J. Brown,
V. P. Knutson,
and
M. K. Haddox.
Multisite phosphorylation of ornithine decarboxylase in transformed macrophages results in increased intracellular enzyme stability, and catalytic effeciency.
J. Biol. Chem.
271:
24945-24953,
1996
31.
Richman, R. A.,
L. E. Underwood,
J. J. W. Van,
and
S. J. Boina.
Synergistic effect of cortisol and growth hormone on hepatic ornithine decarboxylase activity.
Proc. Soc. Exp. Biol. Med.
138:
880-884,
1971.
32.
Rinehart, C. A., Jr.,
and
E. S. Canellakis.
Induction of ornithine decarboxylase activity by insulin and growth factors is mediated by amino acids.
Proc. Natl. Acad. Sci. USA
82:
4365-4368,
1985[Abstract].
33.
Rinehart, C. A., Jr.,
D. Viceps-Madore,
W. F. Fong,
J. G. Oritz,
and
E. S. Canellakis.
The effect of transport system A and N amino acids and nerve and epidermal growth factors on the induction of ornithine decarboxylase activity.
J. Cell. Physiol.
123:
435-441,
1985[Medline].
34.
Seidel, E. R.,
and
D. Ginty.
Apparent post-transcriptional modification of ornithine decarboxylase accounts for its induction in IEC-6 cells in culture.
Digestion
46:
383-389,
1990[Medline].
35.
Seidel, E. R.,
and
V. L. Ragan.
Inhibition by rapamycin of ornithine decarboxylase and epithelial cell proliferation in intestinal IEC-6 cells in culture.
Br. J. Pharmacol.
120:
571-574,
1997[Abstract].
36.
Sertich, G. J.,
and
A. E. Pegg.
Polyamine administration reduces ornithine decarboxylase activity without affecting its mRNA content.
Biochem. Biophys. Res. Commun.
143:
424-430,
1987[Medline].
37.
Shantz, L. M.,
R.-H. Hu,
and
A. E. Pegg.
Regulation of ornithine decarboxylase in a transformed cell line that overexpress translation initiation factor eIF-4E.
Cancer Res.
56:
3265-3269,
1996[Abstract].
38.
Solano, F.,
R. Penafiel,
M. E. Solano,
and
J. A. Lozano.
Equilibrium between active and inactive forms of rat liver ornithine decarboxylase mediated by L-ornithine and salts.
FEBS Lett.
190:
324-328,
1985[Medline].
39.
Stjernborg, L.,
O. Heby,
I. Holm,
and
L. Persson.
On the translation control of ornithine decarboxylase expression by polyamines.
Biochim. Biophys. Acta
1090:
188-194,
1991[Medline].
40.
Tabor, C. W.,
and
H. Tabor.
Polyamines.
Annu. Rev. Biochem.
53:
749-790,
1984[Medline].
41.
Tobias, K. E.,
and
C. Kahana.
Intersubunit location of the active site of mammalian ornithine decarboxylase as determined by hybridization of site-directed mutants.
Biochemistry
32:
5842-5847,
1993[Medline].
42.
Viceps-Madore, D.,
K. Y. Chen,
H. R. Tsou,
and
E. S. Canellakis.
Studies on the role of protein synthesis and of sodium on the regulation of ornithine decarboxylase activity.
Biochim. Biophys. Acta
717:
305-315,
1982[Medline].
43.
Wagner, A. J.,
C. Meyer,
L. A. Laimins,
and
N. Hay.
c-Myc induces the expression and activity of ornithine decarboxylase.
Cell Growth Differ.
4:
879-883,
1993[Abstract].
44.
Wallon, U. M.,
L. Persson,
and
O. Haby.
Regulation of ornithine decarboxylase during cell growth. Changes in the stability and translatability of mRNA, and in the turnover of the protein.
Mol. Cell. Biochem.
146:
39-44,
1995[Medline].
45.
Wang, J.-Y.,
and
L. R. Johnson.
Polyamines and ornithine decarboxylase during repair of duodenal mucosa after stress in rats.
Gastroenterology
100:
333-343,
1991[Medline].
46.
Wang, J.-Y.,
and
L. R. Johnson.
Role of ornithine decarboxylase in the repair process of gastric mucosal stress ulcers.
Am. J. Physiol.
258 (Gastrointest. Liver Physiol. 21):
G78-G85,
1990
47.
Wang, J.-Y.,
S. A. McCormack,
M. J. Viar,
H. Wang,
C.-Y. 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
48.
Wang, J.-Y.,
M. Viar,
P. Blanner,
and
L. R. Johnson.
Expression of the ornithine decarboxylase gene in response to asparagine in intestinal epithelial cells.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G164-G171,
1996
49.
Zimmerman, B. J.,
S. A. McCormack,
M. Israel,
and
L. R. Johnson.
Polyamine-mediated cell migration and growth: structural requirement for diamine analogues to substitute for putrescine.
Cell. Pharmacol.
2:
109-113,
1995.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Visit Other APS Journals Online |