Effect of gastrin on differentiation of rat intestinal epithelial cells in vitro

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.

Key Words: $[Keywords]

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 1.0 × 10⁵ 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 2000 μL, PBS 490 μL and 10 μL pentagastrin solution were replaced to make a final concentration of pentagastrin 250 μg·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 (GenBank^TM accession number M98454) as follows: coding strand primer: 5’-ATGCCCAAGTCAAAGGCTCTCTCAACATCAC-3’, noncoding strand primer: 5’-TGCAACAGTCGCTGGACATCACAGG-3’[10]. The reference primers (Seagon, Shanghai, China) were according to sequences of rat β-actin gene (GenBank^TM accession number AB028846) as follows: coding strand primer: 5’-TTCCAGCCTTCCTTCCTGG-3’, noncoding strand primer: 5’-TTGCGCTCAGGAGGAGCAAT-3’. 2 µL 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 β-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 (Figure 1).

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.

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).

Figure 2 Ultrastructural changes of IEC-6 cells. a: Gastrin-treated cells (5000×, bar = 1 μm) showed columnar structures(the nuclei were shown by open arrows) with numerous microvilli on the edge (solid arrows). b: Gastrin-treated cells (12000×, bar = 500 nm)developed numerous microvilli (open arrows) and lots of endocytic vesicles appeared under the apical membrane (solid arrows). c: Control cells (5000×, bar = 1 μm) 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 (12000×, bar = 500 nm).

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 RT-PCR products from IEC-6 cells on agarose electrophoresis. a: Marker (brighter band: 500 bp), b: Control, c: Gastrin treated cells, d: β-actin(Control), e: β-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)-β1[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 11β-hydroxysteroid dehydrogenase (11β-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 fund amentally 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.