Laboratory of Food Biochemistry, Department of Bioscience and Chemistry, Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan
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ABSTRACT |
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Gene expression of activin, activin receptors, and
follistatin was investigated in vivo and in vitro using
semiquantitative RT-PCR in intestinal epithelial cells. Rat jejunum and
the intestinal epithelial cell line IEC-6 expressed mRNA encoding the
A-subunit of activin,
-subunit of inhibin, activin receptors IB
and IIA, and follistatin. An epithelial cell isolation study focused
along the crypt-villus axis in rat jejunum showed that
A mRNA levels were eight- to tenfold higher in villus cells than in crypt cells. Immunohistochemistry revealed the expression of activin A in upper villus cells. The human intestinal cell line Caco-2 was used as a
differentiation model of enterocytes. Four- to fivefold induction of
A mRNA was observed in postconfluent Caco-2 cells grown on filter
but not in those cells grown on plastic. In contrast, follistatin mRNA
was seen to be reduced after reaching confluence. Exogenous activin A
dose-dependently suppressed the proliferation and stimulated the
expression of apolipoprotein A-IV gene, a differentiation marker, in
IEC-6 cells. These results suggest that the activin system is involved
in the regulation of such cellular functions as proliferation and
differentiation in intestinal epithelial cells.
IEC-6; Caco-2; RT-PCR; crypt-villus axis; differentiation
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INTRODUCTION |
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ACTIVINS, WHICH ARE MEMBERS of the transforming growth
factor- (TGF-
) superfamily, have been shown to regulate a number of different cell functions, including cell proliferation and differentiation (for review, see Refs. 16 and 20). These molecules are
synthesized as either a single homo- or heterodimer of two highly
related
-subunits (
A and
B), resulting in three different isoforms of activins: activin A (
A
A), activin B (
B
B), and activin AB (
A
B) (18, 33). Additional isoforms may arise from
putative activin
C-,
D-, and
E-chains, which have recently been cloned (7, 13, 24). In addition, the
A- and
B-subunits also
form heterodimers with another dissimilar
-subunit, generating inhibin A (
A) and inhibin B (
B) (20). Activins and inhibins act as functional antagonists in some cell systems (18, 33). Activins
bind to binary cell surface receptors, which are those receptors
composed of two single membrane-spanning serine-threonine kinases
designated type I and type II, called Act RI and Act RII, respectively
(for review, see Ref. 21). Still another type of activin-binding
protein is known as follistatin (for review, see Ref. 22). Follistatin,
a family of proteins generated by alternative splicing and proteolytic
processing, has a specific and high affinity for activin, whose effect
it neutralizes in a variety of systems. Thus the biological function of
activins is regulated in concert with its receptors, antagonists
(inhibins), and neutralizing binding proteins (follistatins).
Despite a number of studies investigating the biology of activins,
studies concerning their expression and function in the intestine are
limited. Kawamura et al. (15) demonstrated that human embryonic
intestinal epithelial cell line FHs74Int expressed mRNA encoding the
A-subunit of activin and released bioactive activin A. More
recently, Hubner et al. (14) reported the increased expression of the
A-subunit of activin in surgical specimens from the intestine of
patients with inflammatory bowel disease, suggesting an important role
of activin in inflammatory processes of the intestine. The intestinal
epithelium, which is functionally divided into a zone of proliferation
confined to crypts and a zone of differentiation situated in the villi,
undergoes continuous renewal throughout the lifespan of an animal (3,
11). The terminally differentiated cells of the epithelium are removed by a process of apoptosis occurring throughout the villus and by
exfoliation at the tip of the villus (9, 11). Thus the renewal process
of the intestinal epithelium occurs along the crypt-villus axis. This
entire sequence of cell proliferation, differentiation, apoptosis, and
exfoliation has been thought to be regulated by tissue-specific
programmed gene expression and by such extrinsic factors as the
presence of nutrients and growth factors (1, 2, 4, 17, 28). In view of
the ability of activins to regulate such fundamental cellular functions
as proliferation, differentiation, and apoptosis in diverse cell systems, it is believed that they play a role in the maintenance of
homeostasis in the intestinal epithelium.
The aim of the present study was twofold. First, we attempted to
demonstrate gene expression of activin subunits, activin receptors, and
follistatin in intestinal epithelial cells while, secondly, trying to
obtain information relevant to the association of activins with
proliferation and/or differentiation of epithelial cells. In the
present study, we demonstrated the expression of mRNA encoding the
A-subunit of activin,
-subunit of inhibin, Act RIB, Act RIIA, and
follistatin in the rat jejunum, the intestinal epithelial cell line
IEC-6, and the human colon adenocarcinoma cell line Caco-2. In
addition, we observed that
A mRNA was expressed more abundantly in
villus cells than in crypt cells and that activin A protein was
expressed in upper villus cells, suggesting that expression of activin
A is associated with a termination of proliferation, initiation, and/or
a maintenance of differentiation of intestinal epithelial cells. It was
also shown that gene expression of the
A-subunit of activin was
coupled with cellular differentiation in Caco-2 intestinal cells, which
have been known to differentiate after reaching confluence to form an
epithelium that shares many characteristics of mature small intestinal
mucosa in vivo (10, 24, 26, 32). In contrast, we observed that the
follistatin gene was downregulated in differentiated Caco-2 cells.
Furthermore, it was shown that the addition of activin A to the culture
medium suppressed the proliferation and increased the expression of
apolipoprotein A-IV (apo A-IV) gene, a differentiation marker of
enterocytes, in IEC-6 cells. These results suggest that the activin
system is involved in the maintenance of homeostasis in the intestinal epithelium.
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MATERIALS AND METHODS |
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Animals.
Male Wistar rats (Japan SLC, Hamamatsu, Japan) were housed in
individual cages in a temperature-controlled (23 ± 2°C) room with
a dark period from 1900 to 0500. They were allowed free access to water
and to a purified diet consisting of (as wt/wt) 25% casein, 65%
sucrose, 5% corn oil, 4% mineral mixture, and a 1% vitamin mixture
(30). This diet is used as a standard rat diet in our laboratory because we have found that it yields a maximal growth rate.
Rats weighing between 250 and 300 g were anesthetized by an
intraperitoneal injection of Nembutal (pentobarbital sodium 50 mg/kg
body wt; Abbott Laboratories, North Chicago, IL). After laparotomy, a
10-cm portion of the jejunum at 3 cm distal to the ligament of Treitz
was excised and the luminal contents were thoroughly washed with
ice-cold saline. The mucosa was scraped with a glass slide and
immediately plunged into liquid nitrogen. It was then stored at
80°C for RNA isolation. In a separate experiment, a 20-cm
portion of the jejunum was excised and was subjected to isolation of
the epithelial cells. Furthermore, a 5-cm segment of the jejunum was
excised, embedded in optimum cutting temperature compound
(Miles Scientific, Elkhart, IN), frozen in liquid nitrogen, and stored
at
80°C for immunohistochemistry.
Intestinal cell isolation. Epithelial cells were differentially isolated from the jejunual segments using the distended intestinal sac method described by Traber et al. (31). Briefly, the jejunal segment was rinsed thoroughly with a washing solution composed of 0.15 M NaCl, 1 mM dithiothreitol (DTT), and 40 µg/ml phenylmethylsulfonyl fluoride (PMSF). The segment was then filled with buffer A, composed of 96 mM NaCl, 27 mM sodium citrate, 1.5 mM KCl, 8 mM KH2PO4, 5.6 mM Na2PO4, and 40 µg/ml PMSF (pH 7.4), and the ends were clamped with hemostats. The filled segment was submerged in 0.15 M NaCl at 37°C for 15 min and then drained, and the solution was then discarded. Next, the segment was filled with buffer B, composed of 109 mM NaCl, 2.4 mM KCl, 1.5 mM KH2PO4, 4.3 mM Na2PO4, 1.5 mM EDTA, 10 mM glucose, 5 mM L-glutamine, 0.5 mM DTT, and 40 µg/ml PMSF (pH 7.4), incubated in 0.15 M NaCl at 37°C for 4 min, and then drained. The drained solution was referred to as fraction 1. This step was repeated for another nine cycles at time intervals of 2, 2, 3, 4, 5, 7, 10, 10, and 10 min, generating fractions 2-10, respectively. Finally, these fractions were combined to yield six new fractions: fraction I from fractions 1 and 2, fraction II from fractions 3 and 4, fraction III from fractions 5 and 6, fraction IV from fractions 7 and 8, fraction V from fraction 9, and fraction VI from fraction 10. Aliquots of each fraction were subjected to determine the alkaline phosphatase (ALP) activity using p-nitrophenyl phosphate as a substrate (35). The protein concentration in each fraction was determined by the method of Lowry et al. (19). The cells from each fraction were collected by pelletting at 100 g for 5 min at 4°C, washed once with PBS, and then immediately subjected to isolation of RNA. All solutions used were preoxygenized with 95% O2-5% CO2 and warmed to 37°C.
Immunohistochemistry. To detect the activin A in rat jejunum, immunohistochemistry with catalyzed signal amplification was carried out using a TSA-Indirect kit (NEN Life Science Products, Boston, MA). Frozen sections (6 µm) were prepared with a cryostat, thawed onto glass slides, fixed with 4% (wt/vol) paraformaldehyde in PBS for 10 min, and then washed with TNT buffer (0.1 M Tris · HCl, pH 7.5, 0.15 M NaCl, and 0.05% Tween 20) three times. Sections were incubated with 3% H2O2 in methanol for 10 min to inhibit endogenous peroxidase, washed with TNT buffer three times, and then blocked with TNB blocking buffer [0.1 M Tris · HCl, pH 7.5, 0.15 M NaCl, and 0.5% (wt/vol) blocking reagent (supplied in kit)] for 30 min. Samples were then incubated with primary antibody (anti-activin A monoclonal antibody, IgM class; a gift from Dr. Yuzuru Eto, Ajinomoto, Kawasaki, Japan) at 4°C for 24 h. After washing with TNT buffer three times, secondary antibody (peroxidase-conjugated goat anti-mouse IgM; Biosource, Camarillo, CA) was added for 30 min, followed by washing with TNT buffer three times. Sections were incubated with biotinyl tyramide solution (supplied in kit) for 10 min and then washed with TNT buffer. Samples were incubated with peroxidase-conjugated streptavidin (supplied in kit) for 30 min and then visualized with diaminobenzidine solution (0.5 mg/ml) and 0.05% H2O2 for 5 min. The slides were rinsed with distilled water, counterstained with hematoxylin, dehydrated, air dried, and mounted. The negative control slides were treated with nonspecific mouse IgM.
Cell culture. IEC-6 and Caco-2 cells were obtained from the American Type Culture Collection at passages 13 and 18, respectively. Cells were maintained in Falcon 75-cm2 T-flasks (Nippon Becton Dickinson, Tokyo, Japan) in a standard culture medium at 37°C in a humidified atmosphere of 5% CO2-95% air. The standard culture medium for IEC-6 cells contained DMEM supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 25 mM glucose, 1× nonessential amino acids (from 100× liquid; GIBCO BRL, Tokyo, Japan), 5 mg/l insulin, 100,000 U/l penicillin, 100 mg/l streptomycin, and 50 mg/l gentamycin. The standard culture medium for Caco-2 cells was the same as that for IEC-6 cells except that the medium contained 20% FBS and no insulin. Media were replaced every two days or every day, depending on harvest times and degree of confluence.
For experiments in IEC-6 cells, the cells at 10 days postconfluence were harvested for isolation of RNA. In the case of Caco-2 cells, subconfluent cells were plated onto Falcon six-well plastic plates (Nippon Becton Dickinson) or Falcon 23.4-mm cell-culture inserts (pore size 0.45 µm; Nippon Becton Dickinson) in six-well plastic plates at initial densities of 0.5 × 106 cells/well. The cells were cultured in standard medium and harvested at preconfluence and at 1, 5, 10, 15, and 20 days postconfluence for isolation of RNA. In separate experiments, preconfluent IEC-6 cells were cultured with the standard medium containing graded concentrations of recombinant human activin A (1, 10, 100, and 500 ng/ml; a gift from Dr. Yuzuru Eto) or 1 ng/ml of human TGF-Isolation and analysis of RNA.
Total RNA was isolated from jejunal mucosa, jejunal epithelial cells,
and cell lines using Isogen (Nippon Gene, Tokyo, Japan) according to
the manufacturer's protocol. Total RNA samples were treated with DNase
RQ1 (Promega, Madison, WI) to remove any genomic DNA. Next, 5 µg of
total RNA was annealed with 0.5 µg of oligo(dT)12-18 primer (GIBCO BRL) at 70°C for 10 min, and first-strand cDNA was then synthesized in 20 µl of solution containing 50 mM
Tris · HCl, pH 8.3, 75 mM KCl, 3 mM
MgCl2, 10 mM DTT, 0.5 mM dNTP, 25 units RNase inhibitor,
and 200 units Moloney murine leukemia virus RTase (GIBCO
BRL) at 42°C for 50 min, followed by RNA digestion with RNase H
(GIBCO BRL). The first-strand cDNA sample (0.5 µl) was added to 50 µl of a PCR reaction mixture containing 0.5 µM gene-specific
primers (Table 1), 2 mM
MgCl2, 0.2 mM dNTP, and 1.25 units EX-Taq polymerase
(Takara, Otsu, Japan). Each cycle of PCR included 1 min of denaturation
at 94°C, 1 min of primer annealing at different temperatures for
each primer pair, and 2 min of extension at 72°C. For semi
quantitative PCR, the kinetics of amplification was studied for each
combination of primers in preliminary experiments, and PCR was
performed at an exponential range.
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Statistical analysis. In Figs. 2, 4, and 5, results are expressed as means ± SE. To compare the mean values, Tukey-Kramer HSD was applied. The statistical calculations were carried out using JMP computer software (SAS Institute). Differences were considered significant if P < 0.05.
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RESULTS |
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Expression of mRNA encoding A-subunit of activin,
-subunit of
inhibin, Act RIB, Act RIIA, and follistatin was demonstrated using
RT-PCR and further by Southern blot hybridization with each inner
oligonucleotide probe in the rat jejunum and the rat intestinal epithelial cell line IEC-6. Figure 1 shows
the RT-PCR products separated on 2% agarose gel and their
corresponding Southern blots. In both rat jejunum and IEC-6 cells, the
size of each product was consistent with the predicted size. Under
stringent conditions, each individual band was detected by Southern
blot hybridization of the RT-PCR products with each inner
oligonucleotide probe. In the case of follistatin, a second band of
~770 bp was detected.
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The intestinal epithelial cells were differentially isolated from the
rat jejunum to investigate the expression pattern of each gene along
the crypt-villus axis in the small intestine. Given that we previously
demonstrated by in situ hybridization that apo A-IV mRNA expression
occurs in the epithelial cells located at the upper part of the villus
with no expression in the crypt region in the small intestine (29) and
that polymeric immunoglobulin receptor (pIgR) mRNA is primarily
expressed in the epithelial cells on the lower part of the villus and
upper part of the crypt (unpublished data), detection of mRNA for both
apo A-IV and pIgR validated the differential isolation of the cells
along the crypt-villus axis. Semiquantitative RT-PCR followed by
Southern blot hybridization demonstrated that apo A-IV mRNA was
detected abundantly in fractions I-IV but not in
fractions V and VI (Fig.
2A). In marked
contrast, pIgR mRNA levels were higher in fractions V and
VI than in fractions I-IV. In addition, as ALP
activity has long been considered a marker of intestinal cell
differentiation that is expressed predominantly in villus tip cells
(36), activity of this enzyme was measured in each cell fraction. ALP
activity was highest in fraction I and lowest in fraction
VI (Fig. 2A). Similarly, ALP mRNA levels were higher in
fractions I-III than in fractions IV-VI,
suggesting the pretranslational regulation of ALP expression along the
crypt-villus axis of small intestine. Furthermore, a histochemical
analysis of intestinal remnant after isolation of the epithelial cells demonstrated that the lamina propria of the villus cores was left almost completely intact, whereas the majority of epithelial cells were
eliminated (data not shown). These results suggest that epithelial cells were successfully isolated from along the crypt-villus axis with
minimal contamination of cells from the lamina propria. Figure 2B shows that mRNA for the A-subunit of activin was more
abundant in the higher villus fractions (I-III) than in
the lower villus-to-crypt fractions (IV-VI). In contrast,
no marked changes were observed in mRNA for the
-subunit, Act RIIA,
and follistatin (Fig. 2, B and C). Act RIB mRNA levels
were higher in fractions IV-VI than in fractions
I-III.
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To reveal the expression of activin A protein in rat small intestine,
immunohistochemistry was carried out using anti-activin A monoclonal
antibody. Sakai et al. (27) examined the specificity of the antibody by
preabsorption study and demonstrated that the immunostaining of activin
A in rat tibia was completely absorbed by adding an excess
concentration of recombinant human activin A, suggesting the
specificity of the antibody used here. In the present study, as no
positive signal was detected by the conventional immunoperoxidase
staining (data not shown), we attempted to amplify the signal by using
biotinyl tyramide. Figure 3 showed the
representative results of immunohistochemistry with the catalyzed
signal amplification. The signal was clearly seen in the epithelial
cells in the uppermost part of the villus (Fig. 3, A and
B). In contrast, negative control processed with nonspecific
mouse IgM yielded no signal (Fig. 3C).
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To more fully understand the relationship between the activin system
and differentiation in the human intestinal cell line Caco-2, which has
been used as a model for differentiation of intestinal epithelial cells
in vivo, we investigated the time course for the expression of each
gene. Figure 4 shows representative Southern blots of semiquantitative RT-PCR products in Caco-2 cells grown on filter or plastic. Each individual band was detected by
agarose gel electrophoresis of RT-PCR products, and the size of each
product was consistent with the predicted size (data not shown). Apo
A-IV mRNA was detected as a differentiation marker, and the apo A-IV
mRNA levels increased in both plastic- and filter-grown cells after
reaching confluence (Fig. 4A). In addition, the apo A-IV mRNA
was detected earlier in cells grown on filter than those grown on
plastic. The A-subunit mRNA in the cells on plastic showed no
changes in the very low levels observed during the culture period (Fig.
4B). In marked contrast, the
A-subunit mRNA levels in the
cells grown on the filter were drastically increased after reaching
confluence and higher than in those grown on plastic. The
-subunit
mRNA levels were also increased after confluence, and the levels tended
to be higher in cells on filter than in those on plastic throughout the
culture period (Fig. 4C). In contrast, the follistatin mRNA
levels were drastically decreased after reaching confluence in both
filter- and plastic-grown cells (Fig. 4F). The levels of mRNA
for Act RIB and Act RIIA were also decreased after reaching confluence
(Fig. 4, D and E).
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Furthermore, we investigated whether exogenous activin could influence
the proliferation and differentiation of intestinal epithelial cells.
Addition of recombinant human activin A in the medium suppressed the
proliferation (bromodeoxyuridine uptake) of IEC-6 cells in a
dose-dependent manner (Fig. 5A). In
addition, the apo A-IV mRNA levels in IEC-6 cells were significantly
increased by incubating with exogenous activin A (Fig. 5B).
However, these effects of activin A were markedly weaker than those of
TGF-1.
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DISCUSSION |
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Although the mechanism underlying the renewal process in the intestinal
epithelium, which includes cell proliferation, differentiation, and
elimination, has not yet been fully elucidated, the entire sequence is
believed to be regulated by tissue-specific programmed gene expression
and by such extrinsic factors as the presence of nutrients and growth
factors (1, 2, 4, 17, 28). In terms of growth factors, it has been
reported that both epidermal growth factor and TGF- promote cell
proliferation (2). In contrast, TGF-
has been demonstrated to block
mitotic activity (1) and to induce cell differentiation (17). As
activins, which are members of the TGF-
superfamily, have been
shown to regulate various cell functions, including cell proliferation, differentiation, and apoptosis (16, 20), we may conclude that these
molecules modulate the cell renewal process in the intestinal epithelium. However, few reports to date have demonstrated the expression and function of activins in the intestine.
In the present study using Southern blot hybridization of RT-PCR
products, we demonstrated the mRNA expression of the A-subunit of
activin, the
-subunit of inhibin, Act RIB, Act RIIA, and follistatin in rat jejunum and the rat intestinal epithelial cell line IEC-6 (Fig.
1). From these results, we speculated that activins may modulate the
function of intestinal epithelial cells in concert with antagonists
(inhibins), receptors, and neutralizing binding proteins
(follistatins). Initially, undifferentiated proliferative cells in the
small intestine located in the region of the crypts give rise to
differentiated nonproliferative cells that migrate along the length of
the villus, at which point the terminally differentiated cells are
removed by apoptosis and exfoliation. The present study demonstrated
using a differential cell isolation technique in which the
A-subunit
mRNA levels were overtly higher in the epithelial cells located at the
upper part of the villus than those at the crypt region in rat jejunum
(Fig. 2). The
A-subunit constitutes not only activins (A and AB) but
also inhibin A. As we also showed that the mRNA for the
-subunit of
inhibin was expressed in rat jejunum and IEC-6 cells, it is possible
that inhibin A, in addition to activin A, is synthesized in intestinal epithelial cells. However, unlike the
A-subunit, no changes were observed in the
-subunit mRNA levels in the isolated cells along the
crypt-villus axis. Thus it is likely that synthesis of activin A but
not inhibin A is progressively increased as the cells migrate from the
crypt to the villus. In fact, we confirmed in the present study that
the immunoreactive activin A protein was expressed in the epithelial
cells on the uppermost part of the villus (Fig. 3). Absence of an
activin A signal on the midvillus despite the significant expression of
A-subunit mRNA may be due to low sensitivity of the
immunohistochemistry or pretranslational regulation of activin A
synthesis. These results suggest that activin A is associated with a
termination of proliferation, initiation, and/or maintenance of
differentiation in intestinal epithelial cells.
As cells in the human colon carcinoma cell line Caco-2 spontaneously
differentiate after reaching confluence to form an epithelium with many
characteristics of mature small intestinal mucosa in vivo, this cell
line has been used as an experimental model of cell differentiation in
the small intestine (10, 12, 26, 32). In addition, the culture
substratum has been reported to influence differentiation of Caco-2
cells. Wagner et al. (34) demonstrated that apo A-IV protein was
detectable in the culture media earlier with filter-grown cells than it
was with plastic-grown cells, despite similar apo A-IV mRNA levels. In
the present study, the apo A-IV mRNA levels increased after reaching
confluence in both filter- and plastic-grown cells, but the increment
was earlier in filter-grown cells than in plastic-grown cells (Fig.
4A), a finding that is in contrast to those of Wagner et al.
(34). Nevertheless, these results suggest that Caco-2 differentiation may be facilitated when the cells are grown on a filter. Under these
conditions, we observed that the levels of A-subunit mRNA in
filter-grown cells increased drastically after reaching confluence, whereas the levels in plastic-grown cells were extremely low throughout the culture period (Fig. 3). Thus our findings indicated that gene
expression in the
A-subunit was associated with a differentiation of
cells, a finding that is consistent with the results for the expression
pattern of
A-subunit mRNA and activin A protein along the
crypt-villus axis of rat jejunum. However, it remains to be shown why
A-subunit mRNA in plastic-grown cells was not induced postconfluence
despite the fact that apo A-IV mRNA as a differentiation marker was
increased. In contrast to the
A-subunit, the follistatin mRNA levels
decreased after reaching confluence in both plastic- and filter-grown
Caco-2 cells. The reverse pattern of expression for the
A-subunit
and follistatin suggests that higher levels of follistatin in
undifferentiated proliferative cells may suppress the paracrine action
of activin A secreted from differentiated nonproliferative cells. In
the present study, however, we did not reveal the secretion of
bioactive activin A and follistatin in the Caco-2 cells. Therefore,
further studies will be required to establish the role of activin and
follistatin in the proliferation and differentiation of Caco-2 cells.
To investigate the effect of activin A on the cell proliferation and
differentiation of intestinal epithelium, we used in the present study
the IEC-6 cells, a model for proliferative undifferentiated intestinal
epithelial cells. As TGF- has reportedly blocked the mitotic
activity (1) and induced the differentiation (17) of intestinal
epithelial cells, this growth factor was used as a positive control in
the present study. In fact, addition of 1 ng/ml of TGF-
1 to the
culture medium suppressed the proliferation and stimulated the
expression of apo A-IV mRNA in the IEC-6 cells (Fig. 5). Although
exogenous activin A also suppressed the proliferation and induced the
apo A-IV mRNA in a dose-dependent manner, higher doses were required
compared with the case of TGF-
1. Thus cell proliferation and
differentiation in the intestinal epithelium may be controlled more
dominantly by TGF-
than by activin. Further investigations will be
necessary to make clear how TGF-
and activin share biological
functions in the intestine.
Cell cycle regulatory proteins such as cyclin-dependent kinase
inhibitors (CdkIs) have been implicated in the proliferation and
differentiation of intestinal epithelial cells.
p21WAF1/CIP1 is one of the CdkIs, and its mRNA and protein
product have been demonstrated to be expressed in the epithelial cells
on the uppermost region of the crypt and the villus of small intestine
(5, 8, 25). These observations suggest that p21WAF1/CIP1
expression is inversely related to proliferation and directly correlates to differentiation of intestinal epithelial cells. This
speculation has been supported by cultured-cell studies demonstrating that p21WAF1/CIP1 mRNA and protein were increased in Caco-2
cells after reaching confluence (6, 8). In addition, it has been
demonstrated that activin A inhibited the proliferation of Hep G2
hepatoma cells through upregulation of p21WAF1/CIP1
expression (37). Furthermore, Moustakas and Kardassis (23) reported
that Smad proteins, which play an important role in the transduction of
extracellular signals such as TGF- and activin, transactivate the
p21WAF1/CIP1 promoter in Hep G2 cells. Thus it is likely
that the p21WAF1/CIP1 could mediate the regulation of
proliferation and/or differentiation of intestinal epithelial cells by activin.
In summary, the present study demonstrated that the A-subunit of
activin, the
-subunit of inhibin, Act RI, Act RII, and follistatin
genes were expressed in the intestinal epithelial cells and that
activin A expression could be associated with cell differentiation. It
was also shown that follistatin mRNA was expressed more abundantly in
undifferentiated proliferative Caco-2 cells than in differentiated
nonproliferative cells. The present results suggest that the
activin system is involved in the maintenance of homeostasis in the
intestinal epithelium. Further research is underway to determine the
effects of blockade of activin signal transduction on the proliferation
and differentiation of intestinal epithelial cells.
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ACKNOWLEDGEMENTS |
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Our special thanks are due to Dr. Yuzuru Eto for providing the anti-activin A monoclonal antibody and recombinant human activin A.
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FOOTNOTES |
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This work was partly supported by a Grant-in-Aid for Scientific Research (C) from The Ministry of Education, Science, Sports and Culture of Japan.
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: K. Sonoyama, Laboratory of Food Biochemistry, Faculty of Agriculture, Hokkaido Univ., Kita-9, Nishi-9, Kita-ku, Sapporo-shi, 060-8589, Japan (E-mail: ksnym{at}chem.agr.hokudai.ac.jp).
Received 8 February 1999; accepted in final form 1 October 1999.
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