|
Zhou
Wang, Wei-Wen Chen, Ru-Liu Li, Bin Wen, Piwei Institute,
Guangzhou University of TCM, Guangzhou, 510405, Guangdong Province,
China
Jing-Bo Sun, The Second Affiliated Hospital of Guangzhou
University of TCM, Guangzhou, 510120, Guangdong Province, China
Supported by the Major State Basic Research Development
Program of China (973 Program) No.G19990544 and the National Natural
Science Foundation of China, No.39970906
Correspondence to: Dr. Wei-Wen Chen, Piwei Institute,
Guangzhou University of TCM, Guangzhou, 510405, Guangdong Province,
China. chenww@gzhtcm.edu.cn
Telephone: +86-20-36585080
Fax: +86-20-36586563
Received: 2003-03-04
Accepted: 2003-04-03
Abstract
AIM: To investigate the effect of gastrin on differentiation of
IEC-6 cell line in vitro.
METHODS:
IEC-6 cells were incubated with gastrin. On day 7 after treatment,
cell morphology was examined by light microscope, and on day 20, the
cellular ultrastructures were examined by electron microscope. After
exposure to gastrin for 6 hours, villin mRNA was analyzed by reverse
transcription-polymerase chain reaction, and on day 7, the
expression of villin was examined by immunocytochemical analysis
with laser confocal microscope.
RESULTS:
After exposure to gastrin, IEC-6 cells showed differentiated
phenotypes as villas enterocytes and contained an abundance of
plasma, small nuclei with nucleoli, and were arranged regularly.
There were numerous microvilli around edge of the cells, and several
cells showed columnar structures. Villin mRNA expression in
cytoplasm was increased in comparison with control.
CONCLUSION:
Differentiated characteristics of villus enterocytes and phenotypic
changes of rat intestinal epithelial cells (IEC-6) are induced by
gastrin, and the effects of gastrin are correlated to increased
villin expression.
Wang
Z, Chen WW, Li RL, Wen B, Sun JB. Effect of gastrin on
differentiation of rat intestinal epithelial cells in vitro.
World J Gastroenterol 2003;
9(8): 1786-1790
http://www.wjgnet.com/1007-9327/9/1786.asp
INTRODUCTION
Gastrin stimulates cell proliferation in gastric mucosa under
physiological conditions[1]. Studies have demonstrated
that gastrin many increase ornithine decarboxylase activity of IEC-6
cells, cause intracellular polyamine synthesis, and therefore
promote cell proliferation[2,3] and migration[4].
Polyamine has been demonstrated to be closely correlated to
cytoskeleton reconstitution, an important process of cellular
differentiation[5-9], but it is not clear yet whether
gastrin plays roles in differentiation of IEC-6 cells. This study
was to investigate gastrin-induced morphological changes of
intestinal epithelial cell (IEC-6) and the intracellular expression
of villin.
MATERIALS
AND METHODS
Cell culture
IEC-6 cells (ATCC, Rockville, MD) were grown at 37 °C in a 900 mL.l-1
air-100 mL.l-1
CO2 atmosphere in Dulbecco's
modified Eagle's
medium (pH7.2) containing 50 g.l-1
dFBS, 10 mg.l-1
insulin, 50 mg.l-1
gentamycin sulfate, and subcultured once a week. When cultured cells
became confluence, they were dissociated with 0.5 g.l-1
trypsin and 0.2 g.l-1
EDTA, and seeded into 6-well cell culture plates. Pentagastrin
(Sigma. Louis, MO) was dissolved in two or three drops of 300 g.l-1
ammonium hydroxide (sterile), the solution was adjusted to pH 7.5,
and then diluted with medium to 62.5 mg.l-1
before use.
Morphology
Light microscopy Monolayer
of IEC-6 cells was prepared on glass coverslips, which were placed
in 6-well cell culture plates (Corning Glass Works). The cells were
seeded at a concentration of 1.0105 per well, and incubated at 37 °C in a 900 mL.l-1
air-100 mL.l-1
CO2 atmosphere for 24 h. The media containing cDMEM 2 000
mL,
PBS 490 mL
and 10 mL
pentagastrin solution were replaced to make a final concentration of
pentagastrin 250 mg.l-1
in culture. The medium in control group was the same as that in the
treatment group except gastrin. Cells were harvested on day 7 from
the initial treatment. The coverslips were removed and fixed for 15
min at room temperature in 3.5 % paraformaldehyde in PBS, washed
with distilled water, followed by HE staining and examined under
light microscope.
Electron microscopy The
cells were seeded in 6-well cell culture plates with a concentration
of 1.0×105 per well under the same
culture condition as above. Cultured cells were harvested on day 20
from the initial treatment of gastrin, washed with PBS, fixed in 2 %
glutaraldehyde, postfixed in 1 % osmium tetroxide, dehydrated, and
embedded in Epon, and examined under electron microscope.
Villin
expression
mRNA level analysis After
incubated with gastrin for 6 hours, cultured cells were harvested
for extraction of total RNA with RNA TRIzol reagent (Gibco,
Gaithersburg, MD). Isolation was performed according to the
manufacturer's protocols.
The concentration of extracted RNA was determined. RT-PCR kit (Gibco,
Gaithersburg, MD) was used for RT-PCR reaction following the
attached protocol of the product. The primers (Seagon, Shanghai,
China) were synthesized according to sequences of rat villin gene (GenBankTM
accession number M98454) as follows: coding strand primer: 5'-ATG
CCC AAG TCA AAG GCT CTC TCA ACA TCA C-3', noncoding strand primer:
5'-TGC AAC AGT CGC TGG ACA TCA CAG G-3'[10].
The reference primers (Seagon, Shanghai, China) were according to
sequences of rat b-actin
gene (GenBankTM accession number AB028846) as follows: coding strand
primer: 5'-TTC CAG CCT TCC TTC CTG G-3', noncoding strand primer:
5'-TTG CGC TCA GGA GGA GCA AT-3'. 2 ml
of RT products was added to the
PCR master mix. After incubation at 94 °C for 2 min, reaction
was done for 35 cycles at 94 °C for 60 s, at 55 °C for 60 s, and at 72
°C for 30 s. The
expected cDNA amplification products were 408 bp for villin and 238
bp for b-actin.
After electrophoresis on agarose gel and staining with ethidium
bromide, DNA bands were visualized with an ultraviolet
transilluminator.
Protein level analysis On
day 7 from the initial treatment, cultured cells were fixed for 15
min at room temperature in 3.5 % paraformaldehyde in PBS, and washed
three times. For study of villin expression, the cells were
permeabilized by incubation with 0.2 % Triton X-100 in PBS for 4
min, washed three times with PBS, and then treated with goat serum
for 10 min. The permeabilized cells were incubated with goat
anti-rat antibody with dilution of 1:100 in PBS (Santa Cruz) for 2
hours at room temperature, washed, and then incubated with FITC-conjugated
rabbit anti-goat IgG with dilution of 1:50 in PBS (Sigma. Louis,
MO). The treated cells were visualized under TCS SP confocal laser
scanning microscope (Leica, Heidelberg, Germany).
RESULTS
Effect of gastrin on morphology of IEC-6 cells
Light microscopy Seven-days
after treatment with gastrin, cells were arranged regularly with an
abundance of plasma, and small nuclei with nucleoli. Typically
differentiated cells showed a tendency to form microvilli on the
edge, and remarkable cytoskeleton-like structure, which was similar
to cytoskeleton distribution in well-differentiated enterocytes.
Cells in control group contained sparse plasma, large nuclei without
nucleoli, and were arranged irregularly (Figure1).
Figure 1
Morphology of IEC-6 cells. a:
Gastrin-treated cells(250×) contained an abundance of plasma,
small nuclei with nucleoli, and were arranged regularly. b:
One of gastrin-treated cells (400×) showed the tendency to form microvilli
on the edge(open arrows), and cytoskeleton-like staining in plasma
(solid arrows). c:
Control cells (250×) contained sparse plasma, large nuclei
without nucleoli, and were in irregular arrangement and immature
shape. d: One of
control cells (400×) showed no tendency to form microvilli
on the edge, and nucleus was relatively larger and had no nucleolus.
Figure 2
Ultrastructural changes of IEC-6 cells. a:
Gastrin-treated cells (5 000×, bar=1 mm) showed columnar
structures(the nuclei were shown by open arrows) with numerous
microvilli on the edge (solid arrows). b:
Gastrin-treated cells (12 000×, bar=500 nm)developed numerous
microvilli (open arrows) and lots of endocytic vesicles appeared
under the apical membrane (solid arrows). c:
Control cells (5 000× bar=1 mm) were thin and flat.
Relatively large nuclei (open arrows) and scanty plasma were
observed. d: Only
sparse microvilli (open arrow) and endocytic vesicles (solid arrow)
were seen in control cells (12 000×, bar=500 nm).
Electron
microscopy Twenty
days after 20-day treatment with gastrin, numerous microvilli
appeared on the edge of IEC-6 cells, many endocytic vesicles
occurred under the apical membrane, and columnar structures were
seen in some cells. Control cells were thin and flat, the nuclei
were relatively large with scanty of cytoplasm. Only sparse
microvilli were observed on the edge of control cells, and few
endocytic vesicles were noticed (Figure 2).
Effect of gastrin on villin expression in IEC-6 cells
mRNA level After
exposure to gastrin for 6 hours, villin mRNA expression in gastrin-treated
cells was stronger than that in control cells (Figure 3).
Figure 3(PDF)
RT-PCR products from IEC-6 cells on agarose electrophoresis.
a: Marker (brighter band: 500 bp), b: Control, c: Gastrin treated
cells, d: b-actin(Control),
e: b-actin
(Gastrin).
Protein
level On day 7
after treatment, plenty of cytoplasmic villins were observed
obviously in gastrin-treated cells and few in control cells (Figure
4).
Figure 4 Villin
expression in IEC-6 cells. a:
Gastrin-treated cells (800), b:
Control cells (800).
DISCUSSION
The barrier function of intestine is based on the physiological
renewal or pathological repair of intestinal epithelia. The process
includes proliferation, migration and terminal differentiation of
the crypt cells. Development and differentiation of intestinal
epithelia proceed in at least two distinct steps: the conversion of
a nonepithelial cell to a protoepithelium, followed by a process of
terminal differentiation. Terminal differentiation continues to
occur in adult animals in the intestine[11], and is the
last process. It not only indicates the completion of renewal or
repair and the degree of differentiation, but also determines
whether the new epithelia have physiological functions. There are
two morphological characteristics in differentiated intestinal
epithelia. One is the columnar shape cells with microvilli at the
apical membrane, the other is organization of intestinal epithelial
cells on a basement membrane into multicellular structures.
Intestinal
epithelial cells (IEC-6) have features of undifferentiated small
intestinal crypt cells[12], and are often used as a model
of intestinal mucosal repair and cell differentiation[13,14].
The differentiation of intestinal crypt cell is a complex process,
which is controlled by multiple factors. It has been known that
several genes, such as p38 mitogen-activated protein kinases
(p38MAPK)[15], Cdx gene family[16-20],
pancreatic-duodenal homeobox (Pdxs) gene[21-23],
sucrase-isomaltase (SI) gene[18,24,25], villin[26],
activin[27], provoke cells towards the phenotype of
differentiated villus enterocytes. Some cytokines such as epidermal
growth factor (EGF)[6,7], insulin, insulin-like growth
factor (IGF)-I and II[28,29], transforming growth factor
(TGF)-b1[29],
glucagon-like peptide-2 (GLP-2)[30] also have effects on
the process. Astragalus injection could promote IEC-6 cell
differentiation by inducing ODC activity and polyamine biosynthesis[31].
Moreover,
the interaction between cells or between cells and extracellular
matrix (ECM) also plays an important role in differentiation of
IEC-6 cells[32]. Both humoral and matrix factors from
intestinal mesenchyme are involved in intestinal epithelial
differentiation and these factors appear to be organ specific[33,34].
And in conjunction with cell-cell contact and/or ECM, many
regulatory cytokines such as enteroglucagon, interleukin-2 (IL-2),
fibroblast growth factor (FGF), and EGF family members lead to
specific differentiation signals[35]. Cdx2 gene provokes
pleiotropic effects triggering cells towards the phenotype of
differentiated villus enterocytes, but its expression is also
modulated by basement membrane components[18].
Previous
studies on differentiation of IEC-6 cells have found that laminin
can lead the organization of IEC-6 cells on a basement membrane into
multicellular structures[36], and the down-regulation of
c-jun expression mediated by laminin might result in the event[37].
IEC-6 cell culture on Englebreth-Holm-Swarm (EHS) extracellular
matrix proteins also displays morphological changes, correlated with
loss of nuclear localization of c-myc protein and development of
cell surface alkaline phosphatase (ALP) enzymatic activity[14].
And it has been documented that striking morphological and
functional alterations can be induced by glucocorticoid in IEC-6
cells. These effects are consistent with the activation or
modulation of multiple genes important in physiological functions of
absorptive villous cells[38]. Other data showed
differentiation of IEC-6 cells was associated with upregulation of
11b-hydroxysteroid
dehydrogenase (11b-HSD2)
activity[39]. Members of the Cdx gene family play a
fundamental role in both the establishment of the intestinal
phenotype during development and maintenance of this phenotype via
transcriptional activation of differentiated intestinal genes[40-43].
Our
results showed that significantly morphological changes were
observed in IEC-6 cells treated with gastrin in comparison with
control group. The cells were in regular arrangement. Typically
differentiated cell had an abundance of cytoplasm and a small
nucleus containing nucleolus. There was a tendency to form
microvilli and cytoskeleton-like structures were observed in the
cytoplasm. Twenty-days after treatment of gastrin, a great number of
microvilli appeared on the edges of the cells, and several cells
displayed a simple columnar structure, and were fundamentally
different from adenocarcinoma-like differentiation induced by Cdx1
transfection, which exhibited stratified columnar structure[16].
The absence of a multilayer structure indicated that these cells did
not lose their contact inhibition characteristics, and they were not
tumor cells. The existence of lots of endocytic vesicles as found
under the apical membrane was also a typical feature of terminally
differentiated enterocytes[11,44]. These results
indicated that the cells might have the function of endocytosis as
well as enterocytes. In control cells, only few microvilli were
observed on the edge of cells with few endocytic vesicles.
Villin
is one of the actin-binding proteins which have been reported to
play a major role in the formation of the microvillus core bundle[45].
These proteins are known to modulate the dynamics of the actin
cytoskeleton by mediating the state of actin polymerization and the
spatial arrangement of actin protofilaments[46-49].
Villin may also respond to the apical calcium gradient, fragmenting
actin microfilaments (MFs), and thus locally facilitate actin
remodeling[50], and has a very important role in the
alteration of cell morphology. The villin mRNA was expressed at high
levels in the small intestine, to a lesser degree in the colon, and
was not detected in the brain or liver[51]. The results
indicate that villin is a kind of intestine-specific structure
protein. In HT-29 cells, increase of villin mRNA levels was
consistent with the process of enterocyte differentiation.
Similarly, villin gene expression was induced in Caco-2 cells during
postconfluence differentiation[51]. Immunolocalization
studies on the distribution of the brush border-specific microvillar
protein, villin, in human colonic mucosa indicated that localization
of this protein was disrupted in certain dysplastic and neoplastic
states. Thus, the expression and/or distribution of brush
border-specific proteins such as villin may be useful markers for
defects in the differentiation state of enterocytes[52].
Changes
of cellular morphology and expression of mRNA and protein of villin
in IEC-6 were investigated in order to observe the effects of
gastrin on the differentiation of IEC-6 cells. The results showed
that gastrin could obviously up-regulate villin expression at both
mRNA and protein levels. These results were in consistent with the
morphological alterations of these cells, and indicated that there
was causality between the two events, i.e., gastrin induced
characteristic features of differentiated enterocytes may account
for its up-regulation to villin expression in IEC-6 cells. All these
results indicate that gastrin can promote differentiation of IEC-6
cells, which is correlated to the up-regulation of villin
expression.
ACKNOWLEDGEMENTS
We are grateful to Professor Peixun Wang, Associate Professors
Weiwei Lei and Qin Xu, and Dr. Haibin Wang for their technical
advice and excellent assistance.
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