Department of Surgery, University of Maryland School of Medicine and Baltimore Veterans Affairs Medical Center, Baltimore, Maryland 21201
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ABSTRACT |
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Our previous studies
have shown that inhibition of polyamine biosynthesis increases the
sensitivity of intestinal epithelial cells to growth inhibition induced
by exogenous transforming growth factor- (TGF-
). This study went
further to determine whether expression of the TGF-
receptor genes
is involved in this process. Studies were conducted in the IEC-6 cell
line, derived from rat small intestinal crypt cells. Administration of
-difluoromethylornithine (DFMO), a specific inhibitor of ornithine
decarboxylase (the rate-limiting enzyme for polyamine synthesis), for 4 and 6 days depleted cellular polyamines putrescine, spermidine, and
spermine in IEC-6 cells. Polyamine depletion by DFMO increased levels
of the TGF-
type I receptor (TGF-
RI) mRNA and protein but had no
effect on the TGF-
type II receptor expression. The induced
TGF-
RI expression after polyamine depletion was associated with an
increased sensitivity to growth inhibition induced by exogenous TGF-
but not by somatostatin. Extracellular matrix laminin inhibited IEC-6
cell growth without affecting the TGF-
receptor expression. Laminin
consistently failed to induce the sensitivity of TGF-
-mediated
growth inhibition. In addition, decreasing TGF-
RI expression by
treatment with retinoic acid not only decreased TGF-
-mediated growth
inhibition in normal cells but also prevented the increased sensitivity
to exogenous TGF-
in polyamine-deficient cells. These results
indicate that 1) depletion of cellular polyamines by DFMO
increases expression of the TGF-
RI gene and 2) increased
TGF-
RI expression plays an important role in the process through
which polyamine depletion sensitizes intestinal epithelial cells to
growth inhibition induced by TGF-
.
cell proliferation; transforming growth factor- receptor; laminin; retinoic acid; IEC-6
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INTRODUCTION |
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THE TRANSFORMING
GROWTH factors- (TGF-
) are a family of multifunctional
peptides involved in the regulation of epithelial cell growth and
phenotype (1, 29). There are three distinct but highly
related mammalian isoforms of TGF-
:
1,
2, and
3. TGF-
exert their multiple actions through heteromeric complexes of two types
of transmembrane receptors (type I and type II) with a serine/threonine
kinase domain in their cytoplasmic region (7, 14, 15, 27).
To date, six different TGF-
type I receptors (TGF-
RI) have been
identified in mammals, including T
R-I, ActR-I, ActR-IB, BMPR-IA,
BMPR-IB, and R3 (7, 14, 26). Sizes of the TGF-
RI are
similar to each other (502-532 amino acid residues) and
60-90% amino acid sequence identities in their kinase domains. The TGF-
RI contain a conserved sequence known as the GS domain (also
named type I box) in their cytoplasmic juxtamembrane region (14,
26). The TGF-
RI are more similar to each other than they are
to the known type II receptors (TGF-
RII) and thus form a subgroup of
mammalian type I receptors in the family of receptor serine/threonine kinases.
Exposure of epithelial cells to TGF- leads to inhibition of growth
(1), induction of extracellular matrix protein formation (1, 29), modulation of proteolysis (5), and
stimulation of cell migration (4, 31). To initiate the
signaling of these responses, TGF-
binds directly to the TGF-
RII
that is a constitutive active kinase, after which TGF-
RI is
recruited into the complex (31, 45). The TGF-
RII in the
complex phosphorylates the GS domain of TGF-
RI, which leads to
propagation of further downstream signals (45). Mutational
analyses altering serine and threonine residues in the TGF-
RI GS
domain have indicated that the phosphorylation by TGF-
RII is
indispensable for TGF-
signaling, although its signaling activity
does not appear to depend on the phosphorylation of any particular
serine or threonine residue in the TTSGSGSG sequence of the GS domain
(42, 45).
The intestinal mucosa has the most rapid turnover rate of any tissue in
the body and is continuously renewed from the proliferative zone of
undifferentiated stem cells within the crypts (10). Polyamines spermidine and spermine and their precursor putrescine are
absolutely required for cell proliferation in the crypts of the small
intestinal mucosa, and decreasing the cellular polyamines inhibits
epithelial cell renewal both in vivo (34, 35, 37) and in
vitro (13, 38). We (21) have recently
reported that depletion of cellular polyamines induces the activation
of the TGF- gene through posttranscriptional regulation and that the increased gene product, TGF-
, plays an important role in the process
of growth inhibition after polyamine depletion. Furthermore, we
observed that polyamine-deficient cells were more sensitive to growth
inhibition when they were exposed to exogenous TGF-
(21). However, the mechanism through which polyamine
depletion increases the sensitivity to growth inhibition by TGF-
has
not been demonstrated.
Because intestinal epithelial cells can produce both TGF- receptors
and ligand (1, 29), it is possible that the regulation of
cellular responsiveness relies on the production of active TGF-
and
its presentation to signaling receptors. The present study was designed
to address several questions regarding the involvement of expression of
the TGF-
R genes in the process by which polyamine-deficient
intestinal epithelial cells are more sensitive to exogenous TGF-
.
First, we examined whether depletion of cellular polyamines by
inhibition of ornithine decarboxylase (ODC; the rate-limiting enzyme in
the biosynthesis of polyamines) with
-difluoromethylornithine (DFMO)
increases expression of TGF-
RI and TGF-
RII genes in intestinal
epithelial cells (IEC-6 line). Second, we examined whether inhibition
of TGF-
receptor expression by treatment with retinoic acid (RA)
prevented the increased sensitivity of polyamine-deficient cells to
growth inhibition by TGF-
. Some of these data have been published in
abstract form (25).
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MATERIALS AND METHODS |
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Materials
Disposable culture ware was purchased from Corning Glass Works (Corning, NY). Tissue culture media and dialyzed fetal bovine serum (dFBS) were obtained from GIBCO BRL (Gaithersburg, MD), and biochemicals were from Sigma Chemical (St. Louis, MO). The primary antibody, an affinity-purified rabbit polyclonal antibody against TGF-Methods
Cell culture and general experimental protocol. The IEC-6 cell line was purchased from the American Type Culture Collection at passage 13. The cell line was derived from normal rat intestine and was developed and characterized by Quaroni et al. (24). IEC-6 cells originated from intestinal crypt cells, as judged by morphological and immunologic criteria. They 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 insulin, and 50 µg/ml gentamicin sulfate. Flasks were incubated at 37°C in a humidified atmosphere of 90% air-10% CO2. Stock cells were subcultured once a week at 1:20, and medium was changed three times per week. The cells were restarted from original frozen stock every seven passages. Tests for mycoplasma were routinely negative, and passages 15-20 were used in the experiments. There were no significant changes of biological function and characterization from passages 15 to 20 (38). The general protocol of the experiments and the methods used were similar to those described previously (21). Briefly, IEC-6 cells were plated at 4.25 × 104 cells/cm2 in DMEM plus 5% dFBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate (supplemented DMEM). Cells were incubated in a humidified atmosphere at 37°C in 90% air-10% CO2 (vol/vol) for 24 h; this was followed by a period of different experimental treatments. In the first series of studies, we examined the effect of polyamine depletion on the TGF-RT-PCR. Total RNA was extracted with guanidinium isothiocyanate solution and purified by CsCl density gradient ultracentrifugation as described by Chirgwin et al. (3). Briefly, the cells were washed with D-PBS 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 ~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 lauryl sarcosine, and 5% phenol (added just before use). The addition of 0.1 vol of 3 M sodium acetate and 2.5 vol of ethanol precipitated the purified RNA from aqueous phase in sequence. Final RNA was dissolved in water and estimated from its ultraviolet absorbance at 260 nm using a conversion factor of 40 units.
Ten micrograms of the total RNA were reversely transcribed using a first-strand cDNA synthesis kit (GIBCO BRL) and random hexamers [pd(N)6 primer]. The reaction mixture was incubated for 1 h at 42°C and then heated at 90°C for 5 min to inactivate the reverse transcriptase. The specific sense and antisense primer for TGF-Western immunoblotting analysis.
Cell samples, dissolved in SDS sample buffer, were sonicated for
20 s and centrifuged at 2,000 rpm for 15 min. The supernatant was
boiled for 5 min and then subjected to electrophoresis on 10%
acrylamide gels according to Laemmli (12). Each lane was loaded with 20 µg of protein equivalents. After the transfer of protein to nitrocellulose filters, the filters were incubated overnight
at 4°C in 5% nonfat dry milk in 10× PBS-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 90 min in 1% BSA-PBS-T buffer
containing affinity-purified rabbit polyclonal antibody against
TGF-RI or TGF-
RII protein. The filters were subsequently washed
with 1× PBS-T and incubated for 1 h with anti-rabbit IgG antibody
conjugated to peroxidase by protein cross-linking with 0.2%
glutaraldehyde. After an 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.
Cells were plated at 4.25 × 104/cm2 in
chambered slides and incubated with a medium containing DMEM + 5%
dFBS, 10 µg/ml insulin, and 50 µg/ml gentamicin sulfate. DFMO at a
dose of 5 mM with or without 5 µM spermidine was added as treatment.
The immunofluorescence procedure was carried out according to the
method of Hembrough et al. (9) with minor changes.
Briefly, the cells were washed with D-PBS and incubated with rabbit
anti-TGF-RI antibody at 1:50 dilution for 2 h at 4°C. This
primary antibody recognizes the 55-kDa TGF-
RI in immunoblots of
IEC-6 cell extracts and does not cross-react with other proteins. The
cells were then washed three times with D-PBS, incubated with
anti-rabbit IgG-FITC conjugates (1:100 dilution) for 2 h at 4°C,
rinsed three times again, and fixed in 4% paraformaldehyde. The slides
were mounted with Vectashield mounting medium (Vector Laboratories) and
viewed through a Zeiss confocal microscope (model LSM410).
Electron microscopy. After the cells were grown in the presence or absence of 5 mM DFMO for 4 days, they were washed with D-PBS and then fixed at room temperature in 2.5% glutaraldehyde-3.2% paraformaldehyde buffered with 0.1 M sodium cacodylate (pH 7.4). Cells were postfixed in 2% osmium tetroxide in the same buffer, dehydrated, and embedded in Epon. Ultrathin sections were examined in an electron microscope.
HPLC analysis of cellular polyamines.
The cellular polyamine content was determined as previously described
(38). Briefly, after the cells were washed three times with ice-cold D-PBS, 0.5 M perchloric acid was added, and the cells
were frozen at 80°C until ready for extraction, dansylation, and
HPLC. The standard curve encompassed 0.31-10 µM. Values that fell >25% below the curve were considered not detectable. Protein was
determined by the Bradford method (2). The results are expressed as nanomoles of polyamines per milligram of protein.
Statistics. All data are expressed as means ± SE from six dishes. Autoradiographic and immunofluorescence labeling results were repeated three times. The significance of the difference between means was determined by ANOVA. The level of significance was determined using Dunnett's multiple range test (8).
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RESULTS |
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Effect of Inhibition of Polyamine Synthesis on the TGF-
Receptor Expression
Depletion of cellular polyamines by DFMO resulted in a significant
increase in expression of the TGF-RI gene in IEC-6 cells (Fig.
1). The increase in
mRNA levels for TGF-
RI was noted on day 4 and remained
elevated on day 6 after exposure to DFMO. The levels of
TGF-
RI mRNA in cells exposed to DFMO for 4 and 6 days were ~2.5
times the normal values (without DFMO; Fig. 1, Aa and Ab). Spermidine at a dose of 5 µM given together with DFMO
completely prevented the increased expression of the TGF-
RI gene.
The levels of TGF-
RI mRNA in cells treated with DFMO plus spermidine
were indistinguishable from those in cells grown in control cultures.
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Increased levels of TGF-RI mRNA in cells exposed to DFMO were
paralleled by an increase in TGF-
RI protein levels (Fig. 1, Ba and Bb). TGF-
RI protein levels in cells
exposed to DFMO for 4 and 6 days increased ~2 times over control
values. The TGF-
RI protein concentration was returned to normal
levels when spermidine was given together with DFMO. Putrescine at 10 µM had an effect equal to that of spermidine on the expression of the
TGF-
RI gene when it was added to cultures that contained DFMO (data
not shown). In contrast to TGF-
RI, polyamine depletion did not
induce expression of the TGF-
RII gene in IEC-6 cells. There were no
significant changes in the levels of TGF-
RII mRNA and protein
between control cells and cells exposed to DFMO with or without
spermidine (Fig. 1, A and B).
To extend the finding of increased TGF-RI expression following
polyamine depletion, we further explored the cellular distribution of
TGF-
RI protein in the cells grown in the presence or absence of DFMO
for 4 days with the use of immunohistochemical staining technique. In
control cells, a slight immunostaining for TGF-
RI was observed in a
loosely defined perinuclear area (Fig. 1Ca). Consistent with
our data from Western blot analysis, these immunoreactivities for
TGF-
RI increased markedly after depletion of cellular polyamines by
DFMO (Fig. 1Cb). Increased TGF-
RI was scattered in
punctate foci throughout the cytoplasm. Spermidine given together with DFMO prevented the increased immunostaining for TGF-
RI, and the distribution of TGF-
RI in the cells treated with DFMO plus
spermidine was the same as that in control cells (Fig. 1Cc).
Ultrastructural Changes in Polyamine-Deficient IEC-6 Cells
Normal IEC-6 cells showed a simple monolayer of flat epithelial cells with sparse microvilli (Fig. 2A). In polyamine-deficient cells by treatment with DFMO for 4 days, there was appearance of "membranous lysosomal bodies," which were located throughout the cytoplasm and could be regularly identified in every experiment (Fig. 2, Ba and Bb). These ultrastructural changes were completely prevented by exogenous spermidine given together with DFMO. The ultrastructure in cells grown in the presence of DFMO plus spermidine was similar to that of control cells (data not shown).
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Growth of Polyamine-Deficient Cells in Response to Exogenous
TGF-
|
Polyamine depletion-induced sensitivity to growth inhibition appears to
be specific to exogenous TGF- because the pattern of growth
inhibition induced by somatostatin in DFMO-treated cells was similar to
that of control cells (Fig. 3B). Exposure to somatostatin at
a dose of 250 ng/ml caused no significant inhibition of cell growth in
control cells and DFMO-treated cells. When somatostatin at a dose of
500 ng/ml was given, a significant decrease in cell numbers occurred in
both groups and was ~70% of normal values.
Effect of Laminin on TGF- Receptor Expression and Cell Growth
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Effect of RA on TGF-RI Expression and Cell Growth in IEC-6
Cells
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To determine changes in growth response to exogenous TGF- in the
presence of RA, cells were initially grown in DMEM medium with or
without DFMO for 4 days and then incubated with RA and TGF-
for an
additional 48 h. Figure
7A clearly shows that TGF-
at a dose of 10 ng/ml significantly inhibited normal cell growth (without DFMO), regardless of the presence or absence of RA. However, RA partially but significantly prevented the inhibitory effect of
exogenous TGF-
on cell growth. Consistent with the effect on
TGF-
RI expression, RA at a dose of 2 µM almost completely blocked
the increased sensitivity of polyamine-deficient cells to growth
inhibition induced by exogenous TGF-
(Fig. 7B). When RA
was given together with TGF-
, the inhibitory effect of exogenous TGF-
on cell growth was completely prevented in DFMO-treated cells.
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Effect of Polyamine Depletion on TGF-RI and TGF-
RII
Expression in Other Intestinal Epithelial Cells
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DISCUSSION |
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There is little doubt that intestinal epithelial growth is a
complex process that is controlled at different levels and is modulated
by numerous factors. An adequate supply of polyamines in the dividing
cells within the crypts is absolutely required for small intestinal
epithelial cell renewal (16, 17, 38, 40), but few specific
molecular functions of polyamines in epithelial proliferation have been
defined. The present study clearly shows that inhibition of polyamine
synthesis by DFMO markedly increased expression of TGF-RI mRNA and
protein in IEC-6 cells (Fig. 1). Increased expression of the TGF-
RI
gene was associated with an increase in sensitivity to growth
inhibition induced by exogenous TGF-
but not by somatostatin (Fig.
3). The increased TGF-
RI expression and induced sensitivity to
TGF-
-mediated growth inhibition appear to be specific to polyamine
depletion because laminin-induced growth inhibition did not increase
TGF-
RI expression (Fig. 4) and thus failed to affect cellular
responsiveness to exogenous TGF-
(Fig. 5). Furthermore, decreasing
TGF-
RI expression by treatment with RA not only decreased
TGF-
-mediated growth inhibition in normal cells but also prevented
the increased sensitivity to exogenous TGF-
in polyamine-deficient
cells (Figs. 6 and 7). These findings suggest that expression of the
TGF-
RI gene is highly regulated by cellular polyamines and that
increased TGF-
RI expression after polyamine depletion may play an
important role in the increased sensitivity to TGF-
-mediated growth inhibition.
Although the importance of epithelial cell renewal in the maintenance
of intestinal mucosal integrity is obvious, understanding the elements
regulating intestinal epithelial growth and proliferation is far
from clear. It has been shown that the TGF- signaling pathway
makes a major contribution to these processes and that TGF-
is a
physiological regulator of normal intestinal epithelial growth
(1, 29). Administration of TGF-
inhibits proliferative activity and promotes the development of differentiated function in
intestinal epithelial cells (1). Loss of TGF-
sensitivity is frequently observed in tumors derived from cells that
are normally sensitive, and the extent of TGF-
resistance commonly
correlates with malignancy (6, 15). The TGF-
resistance
in tumor cells may occur after inactivation of essential components of
the TGF-
signaling pathway (15, 28), through depletion
of the p15INK4b locus (32), or
by increased expression of the MDM2 gene (30).
In intestinal epithelial cells, resistance and sensitivity to growth
inhibition by TGF- are mainly regulated by changes in TGF-
receptor expression. Mulder et al. (19) reported that increased TGF-
sensitivity in chemically mutagenized intestinal epithelial cell clones is associated with increases of 5- to 10-fold in
both receptor numbers per cells and binding to signal-transducing (type
I and II) receptors. The IEC-6, IPEC (porcine jejunal enterocytes), and
RIE-1 (rat intestinal epithelial cells) cell lines, all of which are
nontransformed, are growth inhibited by TGF-
and express TGF-
receptors. The ras-transformed RIE-1 and SW-620 transformed cells are not growth inhibited by TGF-
and demonstrate a marked reduction of TGF-
receptor levels (43). A similar
correlation is also observed in rat hepatocytes (20).
During liver regeneration and the condition of primary culture,
increased expression of TGF-
receptors is associated with an
increased sensitivity to TGF-
-mediated growth inhibition in
hepatocytes. In many other cell types, the TGF-
resistance has been
found to relate to a decrease or absence of the expression of TGF-
RI
(11) and TGF-
RII (39).
The results reported here indicate that expression of the TGF-RI is
implicated in the process by which polyamine depletion increases the
sensitivity to TGF-
-mediated growth inhibition in intestinal
epithelial cells. As can be seen in Fig. 1, exposure to DFMO for 4 and
6 days significantly increased levels of TGF-
RI mRNA, which was
paralleled by an increase in TGF-
RI protein. In contrast, expression
of the TGF-
RII gene was not affected after exposure to DFMO in the
presence or absence of exogenous spermidine. This result is not
surprising, because the TGF-
RII is a constitutive, active kinase
(31, 45). Polyamines may regulate TGF-
RII function
through a mechanism rather than through its mRNA and protein synthesis.
In addition, this increased expression of the TGF-
RI gene in
DFMO-treated cells is related to polyamine depletion rather than to the
nonspecific effect of DFMO because the stimulatory effect of this
compound on TGF-
RI expression was completely prevented by the
addition of exogenous spermidine.
Electron microscopic analysis shows that membranous lysosomal bodies appeared within the cytoplasm in DFMO-treated cells (Fig. 2). Although the significance and mechanisms responsible for these membranous lysosomal bodies are unknown, they are completely prevented when spermidine is given together with DFMO, indicating that these ultrastructural changes also result from polyamine depletion. These findings suggest that polyamines may be required for the maintenance of cellular distribution of membrane and nonmembrane organelles in intestinal epithelial cells. This possibility is supported by our previous findings (36), which indicated that inhibition of polyamine synthesis results in the appearance of many punctate foci of actin-myosin II in the cell interior.
It is interesting and of important biological consequences that the
increased TGF-RI expression following polyamine depletion was
associated with an increased sensitivity to growth inhibition induced
by exogenous TGF-
(Fig. 3). This increased sensitivity results from
a specific correlation of TGF-
ligand with its signaling receptors, because there is no change in the sensitivity of
polyamine-deficient cells to somatostatin-mediated growth
inhibition (Fig. 3B). In addition, the increased TGF-
RI
gene expression is related to polyamine depletion and is not a result
of growth inhibition. Extracellular matrix laminin inhibited
intestinal epithelial growth (44) but failed to induce
TGF-
RI expression (Fig. 4). Laminin consistently did not increase
the sensitivity to TGF-
-mediated growth inhibition in IEC-6 cells
(Fig. 5). These results are consistent with our previous findings that
indicate that cellular polyamines negatively regulate expression of
growth-inhibited genes, including p53, junD, and TGF-
in
IEC-6 cells, and that polyamine depletion activates expression of these
genes (13, 21, 23).
To further determine the significance of increased TGF-RI expression
in the induced sensitivity to TGF-
-mediated growth inhibition, RA
was used to decrease TGF-
RI levels in the presence or absence of
cellular polyamines. Retinoids have been shown to modulate expression
of TGF-
receptors in a variety of tissues, and their effects are
cell type dependent. In bovine endothelial cells, RA treatment
upregulates the expression of TGF-
RI and TGF-
RII
(46). On the other hand, TGF-
receptors are markedly decreased after RA exposure in rat liver epithelial cells
(18). Data presented in Fig. 6 show that administration of
RA not only decreased basal levels of TGF-
RI in normal IEC-6 cells
but also prevented the increased TGF-
RI expression in
polyamine-deficient cells. RA treatment also diminished the inhibitory
effect of TGF-
on normal cell growth and prevented the increased
sensitivity of polyamine-deficient cells to growth inhibition induced
by TGF-
(Fig. 7). Although it is possible that RA may have effects
on other factors involved in the regulation of cell growth, these results support the possibility that the induced TGF-
RI after polyamine depletion may be an important determinant of the increased sensitivity to TGF-
action in intestinal epithelial cells.
Data presented in Fig. 8 clearly show that the increased TGF-RI
expression by polyamine depletion is cell type dependent, because
depletion of cellular polyamines by DFMO did not increase expression of
TGF-
receptor genes in Caco-2 cells. The reasons for the different
responses to polyamine depletion in IEC-6 cells and Caco-2 cells remain
obscure and may be related to the fact that the IEC-6 line is derived
from the normal small intestine and Caco-2 line is from the colon
carcinoma. Although we did not examine the effect of polyamine
depletion on the expression of TGF-
receptors in all types of cells
available from small intestine and colon, the present studies also show
that inhibition of polyamine synthesis did not induce the expression of
TGF-
receptor genes in the HT-29 cell line (data not shown).
The nature of molecular mechanisms that increase the expression of
TGF-RI gene after polyamine depletion in IEC-6 cells remains to be
demonstrated. Polyamines have been shown to play different roles in the
expression of various genes in intestinal epithelial cells. For
example, polyamines stimulate the transcription of protooncogene
c-myc and c-jun but have no effect on these two gene posttranscriptions (22). In contrast, polyamines
destabilize the TGF-
mRNA without affecting its transcription
(21). Data presented in Fig. 1A show that the
increased TGF-
RI protein level was paralleled by a significant
increase in TGF-
RI 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
TGF-
RI is due to an increase in gene transcription or results from
the alteration of mRNA stabilization. Our previous studies have
demonstrated that polyamines regulate growth-promoting genes such as
c-myc and c-jun through the stimulation of gene
transcription (22) but modulate growth-inhibited genes
such as TGF-
and p53 through the control of mRNA degradation
(21). We postulate that the increased mRNA level for
TGF-
RI in polyamine-deficient cells may be regulated
posttranscriptionally. This hypothesis awaits further study.
In summary, these results indicate that cellular polyamines are
implicated in the regulation of TGF-RI gene expression in intestinal
epithelial cells. Inhibition of polyamine synthesis by treatment with
DFMO increases the TGF-
RI expression, which associates with the
increased sensitivity to growth inhibition induced by TGF-
.
Decreasing the TGF-
RI level by RA prevents the increased sensitivity
to exogenous TGF-
in polyamine-deficient cells. These findings
suggest that increased TGF-
RI gene expression plays a critical role
in the mechanism through which polyamine depletion increases the
sensitivity of intestinal epithelial cells to TGF-
-mediated growth inhibition.
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ACKNOWLEDGEMENTS |
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This work was supported by Merit Review Grants from the Department of Veterans Affairs to B. L. Bass and J.-Y. Wang and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45314 and DK-57819 to J.-Y. Wang.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J.-Y. Wang. Dept. of Surgery, Baltimore Veterans Affairs Medical Center, 10 N. Greene St., Baltimore, MD 21201 (E-mail: jwang{at}smail.umaryland.edu).
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. Section 1734 solely to indicate this fact.
Received 13 January 2000; accepted in final form 24 April 2000.
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