Transforming growth factor-beta inhibits proliferation and maturation of cultured guinea pig gastric pit cells

Kazuhito Rokutan1, Masahiko Yamada2, Jyunko Torigoe1, and Toshihiko Saito2

1 Department of Nutrition, School of Medicine, The University of Tokushima, Tokushima 770-8503; and 2 Fourth Department of Internal Medicine, Tokyo Medical College, Tokyo 160-0023, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We studied the effects of transforming growth factor-beta 1 (TGF-beta 1) on guinea pig gastric mucous cells, cultured in serum-free conditions. Electron microscopy showed that most cells were pre-pit cells, characterized by the presence of a few secretory granules scattered in the cytoplasm. Epidermal growth factor (EGF) stimulated cell growth, [3H]glucosamine uptake, and accumulation of mucus granules positive for galactose oxidase-Schiff reaction. This EGF-induced maturation into pit cells was confirmed morphologically by the appearance of uniformly dense ovoid or spherical mucus granules packed in the ectoplasm. Western blotting with an antiphosphotyrosine antibody showed that TGF-beta 1 did not inhibit the EGF-initiated tyrosine phosphorylation of the EGF receptor. Northern blotting with cDNA probes for c-fos and c-myc demonstrated that TGF-beta 1 did not affect the EGF-induced expression of the transcripts. However, TGF-beta 1-treated cells did not replicate and remained in an immature stage, even in the presence of EGF, suggesting a potential role of TGF-beta 1 in the regulation of proliferation and differentiation of a pit cell lineage in vivo.

serum-free culture; pit cell lineage; proliferation and differentiation; mucin synthesis; intracellular signals

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

GASTRIC FUNDIC GLANDS have a complex organization of several types of epithelial cells, including pit cells, parietal cells, neck cells, chief cells, and a variety of enteroendocrine cells. These functionally active cells come from multipotent stem cells that are found in the isthmus (19), and filiation and kinetics of the matured cells have been intensively studied in experimental animals. Using the combined techniques of tritiated thymidine labeling and electron microscopy, Karam and Leblond (18-22) identified 11 cell types in the zymogenic zone of the mouse stomach, and they revealed the dynamics of these cells. Recently, transgenic mice have been introduced to study the mechanisms of cell lineage-specific and differentiation-dependent patterns of gene expression in the gastric units (25, 35, 36).

Primary cultures of gastric epithelial cells from rats (40), dogs (6-8), rabbits (39), and guinea pigs (31, 37, 38) have been used to study interactions between distinct growth factors and gastric epithelial cells, and several growth factors, including epidermal growth factor (EGF), transforming growth factor-alpha (TGF-alpha ), hepatocyte growth factor, insulin, and insulin-like growth factor I (IGF-I), have been shown to stimulate proliferation of gastric epithelial cells in those system. Primary cultures of guinea pig gastric epithelial cells usually require the presence of a high concentration of FCS (15, 33, 34). In this case, cultured cells rapidly formed monolayers within 2-3 days, and a majority of the cells (90%) were identified as pit cells or surface epithelial cells (pit top cells) (15). In a previous study (31), we developed a serum-free culture of guinea pig gastric epithelial cells for studying the combined actions of EGF and insulin. In the present experiments, we further characterized this serum-free culture system.

The cells maintained in our serum-free culture system did not exhibit mitotic activity, and electron microscopy showed that a great majority of the cells were in a pre-pit cell stage. EGF stimulated cell growth, and at the same time, EGF-treated cells matured into pit cells that contained large mucus granules positive for galactose oxidase-Schiff (GOS) reaction, suggesting that this culture system may be an excellent model to study the processes of maturation of a pit cell lineage. Using this system, we also studied the effects of TGF-beta on a pit cell lineage. TGF-beta exerts multiple biological actions, such as inhibition of epithelial cell proliferation, modulation of cell migration, and stimulation of extracellular matrix production (4, 26, 28). TGF-beta is produced in gastric mucosa (30) and has been suggested to play an important role in the restitution of injured gastric mucosa (42). However, the effects of TGF-beta on the proliferation and differentiation of gastric epithelial cells have not been studied in detail. We report here that TGF-beta 1 completely inhibits the EGF-induced proliferation and maturation of a pit cell lineage cultured in serum-free conditions.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reagents and media. DMEM, Ham's F-12, and collagen type I were purchased from Flow Laboratories (McLean, VA). RPMI 1640 and MEM were obtained from Nissui Pharmaceutical (Tokyo, Japan). Bovine transferrin and sodium selenite were purchased from GIBCO BRL (Gaithersburg, MD). EGF from mouse submaxillary glands (receptor grade) and human recombinant TGF-beta 1 were purchased from Collaborative Research (Bedford, MA) and King Brewing (Tokyo, Japan), respectively. From American Type Culture Collection (ATCC) (Rockville, MD), we purchased mouse genomic DNAs for c-fos (ATCC no. 41041) and c-myc (ATCC no. 41029) and a cDNA for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ATCC no. 57090). An enhanced chemiluminescence Western blotting detection system, [alpha -32P]dCTP (>3,000 Ci/mmol), hybridization buffer (Rapid-Hyb buffer), and a random primer kit were purchased from Amersham (Tokyo, Japan). Monoclonal antibody specific for phosphotyrosine (clone Py54) was obtained from Oncogene Science (Uniondale, NY). BSA (fraction V) was from Miles Laboratories (Kankakee, IL).

Preparation and culture of gastric mucosal cells. Gastric mucosal cells were aseptically isolated from male guinea pigs weighing ~250 g as described previously (33). The isolated cells were suspended in DMEM/Ham's F-12 (1:1) mixture, containing 15 mM HEPES, 0.2% BSA, 10 µg/ml transferrin, and 2.5 ng/ml sodium selenite. The cells were cultured in 24-well culture plates (collagen type I-coated product from Corning, Corning, NY) or in 35-, 60-, and 100-mm-diameter culture dishes. These culture dishes were coated with collagen type I before use.

Cell growth was analyzed by counting the number of cells with a hemacytometer and by measuring incorporation of [3H]thymidine, as described previously (31).

Histological studies. Glass coverslips were coated with collagen type I and were placed in 35-mm-diameter culture dishes. Isolated cells from guinea pig fundic glands were cultured on the collagen-coated coverslips in the culture dishes for 2 days under the serum-free conditions. Nonadherent cells were removed by washing with MEM. The attached cells on a glass coverslip were untreated or treated with 20 nM EGF and/or 1 nM TGF-beta 1 for 2 days and were subjected to light microscopic examinations. Pit cells and mucous neck cells were distinguished by the reactivity of their mucin granules to GOS or paradoxical concanavalin A (PCS) stainings (23). Parietal cells were identified by immunocytochemical analysis with a monoclonal antibody against the beta -subunit of gastric H+-K+-ATPase, in addition to their fine eosinophilic staining after hematoxylin-eosin staining, as described previously (15).

For examination by transmission electron microscopy, cultured cells in collagen-coated dishes were fixed in 1.25% glutaraldehyde in PBS (pH 7.4) for 20 min at 4°C. After cells were washed with PBS, the fixed cells were postfixed with 1% osmium tetroxide for 1 h at 4°C, dehydrated in a graded series of acetone, and then embedded in Epon. Ultrathin sections were viewed with a Hitachi HU-12 electron microscope.

Detection of phosphotyrosine-containing protein. Isolated cells (1-1.5 × 106) were cultured for 2 days in the serum-free medium in collagen-coated 35-mm-diameter culture dishes. Floating cells were removed by washing with MEM, and the attached cells were incubated for 1 h in the serum-free medium. After exposure of the cells to 20 nM EGF and/or 1 nM TGF-beta 1 for the indicated times, whole cell proteins were extracted and subjected to Western blot analysis with a monoclonal antibody against phosphotyrosine, as previously described (31).

Northern blot analysis. Isolated cells (1 × 107) were cultured in the serum-free medium in collagen-coated 100-mm-diameter culture dishes. Attached cells were treated with 20 nM EGF and/or 1 nM TGF-beta 1 for the indicated times. Total RNA was prepared from the cells with an acid guanidinium thiocyanate-phenol-chloroform mixture (9). Samples of 20 µg of RNA were separated on a 1% agarose gel, blotted, and ultraviolet-crosslinked to a nylon membrane filter (Hybond-N-plus, Amersham). After prehybridization, the filter was hybridized with a cDNA probe for c-fos, c-myc, or GAPDH. A 1.0-kbp Xba I-Sst I fragment of the mouse c-fos genomic DNA and a 1.2-kbp Sst I-Hind III fragment of the mouse c-myc DNA were used to detect the levels of c-fos and c-myc mRNAs. Northern hybridization with the cDNA probes of c-fos and c-myc was performed overnight at 48°C in rapid hybridization buffer (Amersham), containing 40% formamide for c-fos and GAPDH mRNAs or 20% formamide for c-myc mRNA. The probes were prelabeled with [alpha -32P]dCTP by a random primer kit. Membranes were washed once in 2× SSC (1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) containing 0.1% SDS, followed by two washes in 0.2× SSC containing 0.1% SDS. Washing was carried out at 60-65°C. Membranes were exposed to Kodak X-ray films for the appropriate times.

Measurement of incorporation of [3H]glucosamine into cells. After cells were cultured in the serum-free medium for 2 days in collagen-coated 35-mm-diameter dishes, floating cells were removed by washing with PBS, and the remaining cells were incubated overnight in low-glucose DMEM, containing 15 mM HEPES, 0.2% BSA, 10 µg/ml transferrin, and 2.5 ng/ml sodium selenite. These cells were stimulated by 20 nM EGF and/or 1 nM TGF-beta 1. [3H]glucosamine (1 µCi/ml) was added to the cells at the same time or 24 h later. After incubation for 24 h with [3H]glucosamine in the presence or absence of the factors, the medium was removed and the cells were washed with PBS. The cells were lysed by adding 500 µl of 50 mM Tris · HCl buffer (pH, 7.2) containing 2% Triton X-100, and the resultant cell lysate was collected in a microcentrifuge tube. The lysis was completed by passing the lysate through a 27-gauge needle several times. After addition of 500 µl of absolute ethanol, the tubes were placed on ice for 15 min, and precipitated proteins were pelleted by centrifugation at 10,000 g for 20 min at 4°C. After washing three times with 70% ethanol, the precipitate was solubilized in 200 µl of 1 N NaOH and neutralized with 200 µl of 1 N acetic acid. Radioactivity was measured by a liquid scintillation counter and expressed as dpm per milligram of protein.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of TGF-beta 1 on cell growth. When isolated cells (2 × 105 cells), suspended in the serum-free medium, were placed in each well of collagen-coated 24-well culture plates and cultured for 2 days, 0.67 ± 0.9 × 105 cells were attached to each well. After removal of floating cells by washing with MEM, the attached cells were used for studying cell growth. In the serum-free medium, the number of attached cells remained constant for 3 days (Fig. 1A). EGF at 20 nM caused rapid cell growth, doubling the cell number within 24 h. However, treatment with EGF for a longer period did not additionally increase cell number. The EGF-induced proliferation was inhibited by simultaneous addition of TGF-beta 1 in a dose-dependent manner, and TGF-beta 1 at 1 nM completely blocked the growth stimulated by 20 nM EGF (Fig. 1A). TGF-beta 1 itself did not significantly change the cell number during the experimental period (data not shown).


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Fig. 1.   Effects of epidermal growth factor (EGF) and/or transforming growth factor-beta 1 (TGF-beta 1) on cell growth. A: isolated gastric epithelial cells (2 × 105 cells) were placed in each well of collagen-coated 24-well culture plates. After being cultured under serum-free conditions for 2 days, 0.67 ± 0.9 × 105 cells were attached to each well. Floating cells were removed by washing with MEM, and then attached cells were either left untreated (open circle ) or treated with 20 nM EGF (), 20 nM EGF + 0.01 nM TGF-beta 1 (bullet ), 20 nM EGF + 0.1 nM TGF-beta 1 (), or 20 nM EGF + 1 nM TGF-beta 1 (black-triangle) on day 0. After culturing for 24, 48, or 72 h, the no. of cells growing in culture plates was counted, as described in MATERIALS AND METHODS. Values are means ± SD for 18 cultures in 3 different experiments. * Significantly increased compared with cell no. at time 0 (P < 0.05 by Student's t-test). dagger  No. of cells treated with TGF-beta 1 + EGF was significantly decreased compared with that of EGF-treated cells at each time point (P < 0.05 by Student's t-test). B: for measurement of [3H]thymidine uptake, isolated cells (1.5 × 106) were placed in collagen-coated 35-mm-diameter culture dishes. After being cultured for 2 days, attached cells were either left untreated or treated with 20 nM EGF and/or 1 nM TGF-beta 1 (day 0). [3H]thymidine (1 µCi/ml) was added at 0, 24, or 48 h after starting treatment with EGF and/or TGF-beta 1, and the cells were incubated for 24 h. [3H]thymidine uptake during the passing 24-h period was measured, as described previously (31). Values are means ± SD in 4 separate experiments. * Significantly different from untreated cells (P < 0.05 by Student's t-test).

The inhibitory effect of TGF-beta 1 on the EGF-stimulated cell growth was also examined by measuring the incorporation of [3H]thymidine into cells (Fig. 1B). Cells attached to 35-mm-diameter collagen-coated dishes took up a constant amount of [3H]thymidine during 3 days of culture in serum-free conditions. EGF at 20 nM increased the incorporation of [3H]thymidine by two- to threefold within 24 h. Although 1 nM TGF-beta 1 did not change the cell number, it significantly decreased the incorporation of [3H]thymidine on day 3 (48-72 h) compared with that of untreated control cells (Fig. 1B). Similarly, TGF-beta 1 decreased [3H]thymidine uptake by EGF-treated cells to a lower value than that of untreated control cells on day 3.

Effects of TGF-beta 1 on EGF-induced protein tyrosine phosphorylation. To elucidate the mechanism of the inhibitory effect of TGF-beta 1 on EGF-stimulated cell growth, we tested whether TGF-beta 1 affected EGF-dependent intracellular events. As shown in Fig. 2 (lanes 3-5), EGF caused rapid tyrosine phosphorylation of a protein with a molecular mass of 170 kDa. This 170-kDa protein was already identified as the tyrosine-autophosphorylated EGF receptor (31). A strong immunoreactive band with a molecular mass of 68 kDa was BSA, contaminated from the serum-free culture medium. Several other protein bands were identical before stimulation by EGF, but the intensities of these bands were not changed after addition of EGF. TGF-beta 1 did not initiate any protein tyrosine phosphorylation (Fig. 2, lanes 9-11) and did not affect the EGF-induced autophosphorylation of the EGF receptor (Fig. 2, lanes 6-8), suggesting that TGF-beta 1 might affect postreceptor signaling pathways of EGF.


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Fig. 2.   Effects of EGF and/or TGF-beta 1 on protein tyrosine phosphorylation. After being cultured in serum-free conditions in collagen-coated culture plates for 2 days, attached cells were unstimulated (lanes 1 and 2) or stimulated with 20 nM EGF (lanes 3-5), 20 nM EGF + 1 nM TGF-beta 1 (lanes 6-8), or 1 nM TGF-beta 1 (lanes 9-11). Whole cell proteins were extracted, as previously described (33), and an equal amount of protein (30 µg protein/lane) was separated by 8% SDS-PAGE and transferred to a polyvinylidine difluoride membrane. After blocking with 4% milk casein, the membrane was incubated with a monoclonal antibody against phosphotyrosine. Bound antibodies were detected with the use of an enhanced chemiluminescence detection kit. Similar results were obtained in 3 separate experiments. MMSTD, molecular mass standard.

Effect of TGF-beta 1 on EGF-induced expression of c-fos and c-myc mRNAs. Low levels of c-fos and c-myc mRNAs were detected in untreated control cells (Fig. 3). When cultured cells were stimulated by EGF, they rapidly and transiently increased the c-fos mRNA level with a peak at 30-60 min. TGF-beta 1 alone did not cause the c-fos mRNA expression and did not change the EGF-stimulated accumulation of c-fos mRNA (Fig. 3A). EGF also increased c-myc mRNA with a peak at 2 h (Fig. 3B), while TGF-beta 1 did not. Furthermore, TGF-beta 1 did not alter the magnitude and duration of the mRNA expression induced by EGF.


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Fig. 3.   Expression of c-fos (A) and c-myc (B) mRNAs in gastric epithelial cells. Gastric epithelial cells were cultured for 2 days in collagen-coated 60-mm-diameter culture dishes in serum-free conditions. After treatment of attached cells with 20 nM EGF and/or 1 nM TGF-beta 1 for the indicated times, total RNA was extracted, and Northern blot analysis with cDNA probes for c-fos, c-myc, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was performed, as described in MATERIALS AND METHODS. Similar results were obtained in 4 separate experiments.

Cytochemical identification of cultured cells. After cultivation of isolated cells for 2 days in collagen-coated plates under complete FCS-free conditions, a majority of the cells (>80% of total cells) did not contain large mucus granules (Fig. 4A). About 5% of the cells contained gatherings of large mucus granules that were visualized by GOS reaction (Fig. 4A), suggesting that these cells are mature pit cells. Less than 1% of the cells contained PCS-positive granules, which are relatively specific for mucous neck cells (data not shown). Parietal cells were characterized by immunoreactivity for antibody against the beta -subunit of gastric H+-K+-ATPase with 4%-6% of the cultured cells being identified as parietal cells (data not shown). When gastric epithelial cells were cultured in FCS-containing media, ~90% of cells possessed large GOS-positive granules (15). Thus the population of mucin-containing cells was strikingly changed by the presence of FCS, and a majority of the cells, cultured in serum-free conditions, did not have enough mucus granules to be detectable by the GOS reaction.


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Fig. 4.   Galactose oxidase-Schiff (GOS) staining of gastric epithelial cells. Isolated cells were cultured for 2 days on collagen-coated glass coverslips in 35-mm-diameter culture dishes under serum-free conditions. Attached cells were untreated (A) or treated for 2 days with 20 nM EGF (B), 1 nM TGF-beta 1 (C), or 20 nM EGF + 1 nM TGF-beta 1 (D). Cells were subjected to GOS and hematoxylin stainings. Arrows indicate gatherings of GOS-positive mucus granules.

After treatment with EGF, GOS-positive materials gradually increased, and 2 days later, a majority of cells contained large GOS-positive granules (Fig. 4B), suggesting that EGF might stimulate not only proliferation of the mucin-less cells, but also maturation of the cells into pit cells. When cultured cells were treated with TGF-beta 1, GOS-positive granules were not observed (Fig. 4C). It was of interest that TGF-beta 1 completely blocked the EGF-induced accumulation of GOS-positive granules (Fig. 4D). TGF-beta 1 and/or EGF did not increase the population of mucous neck cells, which was confirmed by PCS staining for 2 days after treatment with the two factors (data not shown).

Effect of EGF and/or TGF-beta 1 on [3H]glucosamine uptake. To confirm the cytochemical data, mucin synthesis in the absence or presence of EGF and/or TGF-beta 1 was estimated by measuring the incorporation of [3H]glucosamine into cells (Fig. 5). In untreated control cells, [3H]glucosamine uptake was constant during the experimental period. TGF-beta 1 itself did not change the basal level of the uptake. EGF significantly stimulated the [3H]glucosamine uptake by the mucin-less epithelial cells. This increase was again blocked when TGF-beta 1 was included, supporting the cytochemical study that TGF-beta 1 could block the stimulatory action of EGF on mucin synthesis.


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Fig. 5.   Effects of EGF and/or TGF-beta 1 on [3H]glucosamine uptake by gastric epithelial cells. Isolated cells were cultured for 2 days in collagen-coated 35-mm-diameter dishes under serum-free conditions. Attached cells were untreated or treated with 20 nM EGF and/or 1 nM TGF-beta 1. [3H]glucosamine (1 µCi/ml) was added at the same time or 24 h after addition of EGF and/or TGF-beta 1. After incubation for 24 h, radioactivity incorporated into cells was measured, as described in MATERIALS AND METHODS. Values are means ± SD for 12 cultures in 3 separate experiments. * Significantly different from untreated cells (P < 0.05 by Student's t-test).

Electron microscopy of cultured gastric epithelial cells. Cytochemical studies with GOS and PCS reactions suggested that EGF might stimulate the maturation of progenitor cells into pit cells and that TGF-beta 1 might counterregulate this EGF action. To confirm these EGF and TGF-beta 1 actions, we performed electron microscopy, since the stage of differentiation of a pit cell lineage is well characterized by the appearance of secretory granules (19, 20).

Cytochemical studies showed that GOS-positive granules were seen in a small number of untreated control cells; however, most untreated cells had large nuclei and were characterized by the presence of a few secretory granules scattered in the cytoplasm, as shown in Fig. 6A (cell at right). Granule-free cells were sporadically observed, as seen in Fig. 6A (cell at left). Two days after addition of EGF, the cells exhibited features characteristic of matured pit cells; they possessed uniformly dense ovoid or spherical mucus granules packed in a peripheral area (ectoplasm) (Fig. 6, B and C). Granule size increased up to 1,000 nm. These cells showed a decrease in nucleus size. Large secondary lysosomes were frequently observed, and vacuoles were sometimes seen in the cytoplasm (Fig. 6C). Typical features of TGF-beta 1-treated cells are shown in Fig. 6D. TGF-beta 1-treated cells consisted of a relatively homogenous population, compared with control cells or EGF-treated cells. TGF-beta 1-treated cells had large nuclei, and they seldom contained apparent secretory granules. They also contained dark, granular-like structures, which seemed to be secondary lysosomes rather than secretory granules (Fig. 6, D and E). Thus TGF-beta 1-treated cells exhibited more immature features than those of control cells and EGF-treated cells.


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Fig. 6.   Electron microscopy of cultured gastric epithelial cells. Isolated cells were cultured for 2 days in collagen-coated 35-mm-diameter culture dishes in serum-free conditions, and attached cells were untreated (A) or treated for 2 days with 20 nM EGF (B and C), 1 nM TGF-beta 1 (D), or 20 nM EGF + 1 nM TGF-beta 1 (E). Cells were observed by transmission electron microscopy, as described in MATERIALS AND METHODS. N, nucleus; Ly, lysosome; MV, microvilli; sg, secretory granule.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Gastric surface epithelial cells originate from the granule-free progenitor cells located at the isthmus of the gastric unit (19). During upward migration, granule-free pre-pit cell precursors acquire secretory granules at a pre-pit cell stage, and then secretory granules increase in number and size along with maturation into a final surface epithelial cell stage. The matured cells display morphological and biochemical properties, characterized by their large granules, consisting of two major components, GOS-positive mucin and surface-active phospholipids (6, 12).

Guinea pig gastric epithelial cells, dispersed by protease digestion, rapidly attached to plastic culture dishes and formed monolayers within 2-3 days in the presence of 10% FCS. FCS stimulated rapid cell growth within 24 h, and then the cells gradually lost their mitotic activity when the great majority of the cells acquired large GOS-positive granules characteristic of pit cells (31). A great amount of information on the mechanism of proliferation of gastric mucous epithelial cells has been obtained from primary cultures of gastric epithelial cells (7, 8, 33, 37-39). In those experiments, serum-starved cells were used to examine mitogen responses, since the presence of serum may perturb epithelial cell function and differentiation. FCS was required for adherence of cells to plastic culture plates. Chen et al. (8) established a short-term culture of mucosal replicating cells from canine oxyntic glands, and serum effects were minimized by limited exposure to serum only for the first 12-18 h of culture. However, we noticed that a short-term exposure to FCS (for >6 h) could prime the cells for maturing into mucin-containing cells. Therefore, we completely removed FCS for the entire culture period to permit detailed study on proliferation and maturation of a pit cell lineage by individual growth factors.

When isolated cells were suspended in the serum-free medium and placed in collagen type I-coated dishes, immature cells slowly and selectively attached to the plates. These cells did not replicate in serum-free conditions. Cytochemical and electron microscopic studies showed that a majority of cells (~90%) displayed features characteristic of a pre-pit cell stage. In response to EGF, pre-pit cells rapidly proliferated and then acquired large GOS-positive granules on day 2. At that time, these mature cells did not replicate any more. This cessation of cell growth was not due to contact inhibition in our assay conditions. These findings suggest that the immature cells, maintained in our culture conditions, may already be programmed to mature into pit cells and surface epithelial cells, once they are stimulated by a mitogen, such as EGF. Using a novel cell line derived from the gastric epithelial cells of transgenic mice harboring the temperature-sensitive simian virus 40 T antigen, Konda et al. (24) also showed that gastric mucous cells ceased growing when they exhibited periodic acid-Schiff reaction-positive materials. Thus the present culture system might, at least in part, reemerge the process of maturation of a pit cell lineage in vivo.

With use of primary cultures of gastric mucous epithelial cells from different animals, several growth factors, including EGF, TGF-alpha , hepatocyte growth factor, insulin, and IGF-I, have been shown to stimulate proliferation of gastric epithelial cells (7, 8, 33, 37-39). On the contrary, growth-inhibitory factors have not been studied in detail. We showed that TGF-beta 1 completely inhibited EGF-induced proliferation of gastric epithelial cells in culture. Furthermore, TGF-beta 1 additionally decreased [3H]thymidine uptake below the basal level even in the presence of EGF. TGF-alpha protein is expressed in luminal surface epithelial cells and those lining the gastric pit in normal human stomach (41). It was shown that mature mucus-producing cells in culture produced TGF-alpha (2, 7, 24), while immature pit cells expressed low levels of this factor (24). TGF-alpha has been suggested to play an important role in density-dependent growth by autocrine and/or paracrine mechanisms (7). A majority of the cultured cells in our serum-free conditions were composed of pre-pit cells, and a small proportion of the cells (<5%) appeared to be pit cells, containing uniformly dense spherical or ovoid mucus granules that were detectable by the GOS reaction. Although the cell number and [3H]thymidine uptake by untreated control cells remained constant, our culture system could not completely eliminate the autocrine and/or paracrine regulation of cell growth (i.e., through TGF-alpha ). It is possible to speculate that TGF-beta 1 might block the autocrine and/or paracrine control; therefore, it additionally decreased the [3H]thymidine uptake below the control level.

It was of interest that TGF-beta 1 also inhibited accumulation of GOS-positive granules in the cells stimulated by EGF. TGF-beta has been widely viewed as a growth-stimulatory factor for mesenchymal cells and a growth inhibitor for epithelial cells and is also known as a potent differentiation factor on several types of cells (4, 26, 28). Cytochemical and electron microscopic examinations revealed that the TGF-beta 1-treated cells remained in an immature pre-pit cell stage, suggesting that TGF-beta 1 may act as a potent maturation-inhibitory factor rather than as a differentiation factor for a pre-pit cell lineage. In the stomach, expression, localization, and physiological roles of this factor have not been fully understood. At present, it is doubtful whether TGF-beta actively controls the proliferation and differentiation of a pit cell lineage in normal gastric mucosa, since it was reported that fibroblasts and granulocytes sporadically showed immunoreactivity for proTGF-beta , while glandular epithelial cells were all negative in the normal human stomach (30). However, it was reported that hyperplastic lesions similar to human gastric cystica profunda developed in the gastric glandular mucosa of TGF-beta heterozygous mice (5). Several lines of evidence have suggested that TGF-beta may play an important role in the restitution and healing of injured gastric mucosa (10, 42). TGF-beta simultaneously exerts multiple biological actions, such as increases in the synthesis and deposition of extracellular matrix and modulation of cell migration, and all of these sequential events contribute to wound repair (1, 3). In fact, transient expression was also detected in acute phase gastric ulcer (10). The overexpression of this factor was detected in the mesenchymal cells of fibrous granulation tissue in the stomach and in diffuse-type gastric carcinoma (17, 30). In diffuse-type gastric carcinoma, cancer cells as well as stromal cells (fibroblasts, macrophages, and endothelial cells) expressed abundant proTGF-beta . TGF-beta secreted from the cells has been suggested to promote extensive fibrosis in those lesions, in which TGF-beta may act as a potent growth inhibitor of mucus-secreting cells, as demonstrated in this study.

We also examined the effects of TGF-beta 1 on EGF signaling pathways. Many lines of evidence have suggested that TGF-beta does not inhibit EGF binding to its receptor and early growth factor-induced events (29). This was also in the case in gastric epithelial cells; TGF-beta 1 did not affect the autophosphorylation of the EGF receptor induced by EGF. TGF-beta induces growth inhibition by upregulating cyclin-dependent kinase (CDK) inhibitor p15 in certain epithelial cell lines (13). In addition, the antiproliferative effect of TGF-beta is also mediated by repression of the expression of Cdc25A, a CDK tyrosine phosphatase that activates CDK (16). TGF-beta has been shown to repress c-myc expression, and c-Myc can transcriptionally induce Cdc25A (11), leading to a possibility that the suppression of c-myc expression has been considered to be a central event in the growth-inhibitory response to TGF-beta (32, 16). In our experiments, however, TGF-beta 1 did not affect the duration and magnitude of c-myc mRNA expression after stimulation of cultured gastric epithelial cells by EGF. Yoshimura et al. (43) also reported that the degree of c-fos and c-myc expressions by EGF, insulin, dibutyryl-cAMP, or TGF-beta did not necessary correlate with the effects of these agents on cell proliferation of rabbit gastric epithelial cells in culture. TGF-beta family members signal through two transmembrane serine/threonine kinases known as the type I and type II receptors. Recently, the SMAD [a new word coined from Sma and Mad (Mothers against dpp)] family of signal transducer proteins has been identified to be a component in signal transduction pathway downstream of the serine/threonine kinase receptors (14, 27). Different members of the SMAD family have different roles in signaling, and TGF-beta signaling has not been studied in gastric epithelial cells. Elucidation of TGF-beta signals and its target genes may help to understand the regulatory mechanism of proliferation and differentiation of a pit cell lineage.

    FOOTNOTES

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: K. Rokutan, Dept. of Nutrition, School of Medicine, The Univ. of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.

Received 11 March 1998; accepted in final form 20 May 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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