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Stem Cells 2005;23:433-441 www.StemCells.com
© 2005 AlphaMed Press

Retinoic Acid Stimulates the Dynamics of Mouse Gastric Epithelial Progenitors

Sherif M. Karama, Rony Johna, David H. Alpersb, Abdul Samad Ponerya

a Department of Anatomy, Faculty of Medicine and Health Sciences, United Arab Emirates University, Al-Ain, United Arab Emirates;
b Department of Medicine, Washington University School of Medicine, St. Louis, Missouri, USA

Key Words. Gastric stem cell • Cell dynamics • Pit cell • Zymogenic cell • Vitamin A • Stomach

Correspondence: Sherif M. Karam, M.D., Ph.D., Department of Anatomy, Faculty of Medicine and Health Sciences, UAE University, Al-Ain, P.O.Box17666, United Arab Emirates. Telephone:971-3-703-9493; fax:971-3-767-2033; e-mail: skaram{at}uaeu.ac.ae


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The gastric epithelial progenitors proliferate and undergo bipolar migration associated with their differentiation into pit, parietal, and zymogenic cell lineages. Retinoids have long been known to modulate proliferation and differentiation of various renewing epithelia, and the expression of their receptors has been demonstrated in the gastric mucosa. The aim of this study was to examine the effects of retinoic acid on progenitor cell proliferation and cell lineage formation in the mouse stomach. By using subcutaneously inserted osmotic pumps, mice were continuously infused with all-trans retinoic acid (5 mg/kg per day) for 3 days. To label S-phase cells and their progeny, bromodeoxyuridine was administered for different time intervals. Analysis of gastric mucosal tissues of retinoic acid–treated mice revealed a significant increase in the number of S-phase progenitor cells and an enhancement in the production of their progeny. The life span of pit cells was reduced, and their apoptosis became apparent at the luminal surface. Immunofluoresence probing of pit, parietal and enteroendocrine cell lineages in control and retinoic acid–treated mice showed no significant change in their labeling pattern. However, there was an increase in the labeled gland area of zymogenic cells. In conclusion, 3-day treatment of retinoic acid enhances the proliferation of gastric epithelial progenitors and the dynamics of their progeny.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the mouse stomach, the oxyntic epithelium is organized to form numerous short pits populated by pit and parietal cells producing mucus and acid, respectively. The pits open into long tubular glands lined by mucous neck, parietal, enteroendocrine, and zymogenic cells. The latter secretes pepsinogen and intrinsic factor. Each gland also includes epithelial progenitors anchored in a narrow region next to the pit boundary [1]. Normally, these progenitor cells proliferate actively to reproduce themselves and to differentiate during a spatially well-organized bipolar migration toward the luminal surface and the gland bottom and therefore produce all cells lining the pit-gland unit [2].

Similar epithelial progenitors have been defined recently in the human stomach and also found to be responsible for the continuous production of all cell types in the gastric epithelium [3]. Although it is generally believed that these progenitor cells play an important role during gastric carcinogenesis [4] and, in a transgenic mouse model, their transdifferentiation into neuroendocrine gastric cancer cells has been recently demonstrated [5], little information is available regarding the factors that control the dynamic features of these epithelial progenitors.

Vitamin A or retinoic acid is known to have profound effects on cell proliferation and differentiation in other renewing epithelia. It has been shown to play an important role in epidermal keratinization [6], mammary gland formation [7], and gene expression of tracheobronchial epithelium [8]. Retinoic acid acts through specific receptors that are members of the nuclear steroid receptor superfamily of proteins. These receptors function as ligand-dependent transcription factors that are believed to control cell proliferation and differentiation [9].

In the human stomach, retinoic acid is thought to play a role in gastric cancer prevention and has long been known as a cytoprotective agent against gastric mucosal damage and ulcer formation by mechanisms that are not well understood [10]. Mozsik et al. [11] have shown that the action of retinoic acid depends on intact adrenals and vagal innervation; however, the possibility that it has direct effects on the gastric epithelium was not excluded. Recently, mRNA expression of the retinoid receptors was demonstrated in the gastric mucosa [12, 13]. However, still there is a debate whether retinoic acid can be used as a chemopreventive agent against gastric cancer. Although some investigators found that it prevents progression of atrophic gastritis to gastric cancer [14], others reported that it has no effect on cancer progression and may even increase the risk of other types of cancer, such as bronchogenic carcinoma [15].

This report examines whether retinoic acid has an effect on the proliferation of gastric epithelial progenitors. Also, it provides some insights into a possible role of retinoic acid in the control of differentiation program and turnover of the gastric epithelium.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Continuous Infusion of Mice With Retinoic Acid
The following protocol was approved by the Research Ethics Committee of the Faculty of Medicine and Health Sciences, United Arab Emirates University. Young adult (1- to 2-month-old) C57BL/6J mice were given continuous subcutaneous infusion of all-trans retinoic acid (5 mg/kg body weight per day) for 3 days via Alzet micro-osmotic pumps (Durect Co, Cupertino, CA). The pumps were prepared for implantation according to the manufacturer’s instructions. The mice were anesthetized with ketamine (50 mg/kg), and pumps were inserted subcutaneously via a small incision in the back. Wound clips were used to close the incision, and mice were monitored for 2 hours after implantation and at least twice per day during the infusion period. Each retinoic acid–treated mouse was paired with a sex- and weight-matched littermate, which was given a continuous infusion of vehicle. Mice were killed by an overdose of the anesthetic, and the stomachs were fixed in Bouin’s solution and processed for paraffin embedding.

Cell Proliferation and Migration Studies
In some experiments, retinoic acid–treated and control mice received an intraperitoneal injection of 5-bromo-2'-deoxyuridine (BrdU) (120 mg/kg body weight) 1 hour before euthanasia to label cells in S-phase. To label progeny of S-phase cells and follow their migration pathway, some mice were given, along with retinoic acid, a continuous subcutaneous infusion of BrdU (400 mg/kg body weight per day) for 1 or 3 days via the Alzet osmotic pumps.

Animals were killed by a lethal dose of the anesthetic, and the stomachs were opened, fixed in Bouin’s solution, and processed for paraffin embedding. Sections were incubated with goat polyclonal antibodies specific for BrdU [16]. The secondary antibody used was fluorescein isothiocyanate (FITC)- or peroxidase-conjugated rabbit anti-goat immunoglobulin G (IgG). Sections were counter-stained with tetramethylrhodamine isothiocyanate (TRITC)-conjugated Ulex europaeus agglutinin type 1 lectin (UEA-1) (Sigma) or periodic acid Schiff (PAS) to label pit cells.

In sections from mice given a single BrdU injection, cells with labeled nuclei were counted per longitudinally cut gland. For each mouse, at least fifteen glands were examined in three different sections. Counts are expressed as mean ± standard error of the mean (SE).

Because pit cells are short-lived cells and many would incorporate BrdU during a 3-day infusion, the overall labeling index of pit cells was determined after single injection and continuous infusions of BrdU. A phase-contrast microscope was used to count the number of pit cells with labeled nuclei and the total number of pit cells (with labeled and unlabeled nuclei) per gland. The labeling indices were plotted against time, and the slope of the regression line was used to estimate the turnover rate of pit cells in control and retinoic acid–treated mice, as previously reported [17].

To demonstrate cell migration in sections of BrdU-treated tissues, labeled and unlabeled cells were scored in two equal segments of the pit (low and high) and in three different gland regions (isthmus, neck, and base). Then the labeling indices were estimated in each region or segment. Data were expressed as mean of percent labeling in each region or segment ± SE. Student’s t test was used to compare counts or percent of BrdU-labeled cells in control versus retinoic acid–treated mice.

During cell scoring, the borders of the regions of the gastric glands were defined according to previously reported criteria [1]. Briefly, the pit-isthmus border is taken to be the lower edge of the deepest pit cell; the isthmus-neck border is defined by the upper edge of the most superficial neck cell; the upper edge of the most superficial zymogenic cell represents the beginning of the base region.

Immunofluorescence Labeling of Cell Lineages
To distinguish the different gastric cell lineages, sections were incubated with primary antibodies or lectins as previously reported [18]. The following antibodies were used: mono-clonal anti-H, K-ATPase ß-subunit for parietal cells (Cal-biochem-Novabiochem Co, La Jolla, CA), rabbit polyclonal anti-intrinsic factor antibody against baculovirus-expressed human intrinsic factor for zymogenic cells [19], polyclonal anti-gastrin antibodies (DAKO) for the antral G cells, and polyclonal anti-ghrelin antibodies for various oxyntic enteroendocrine cells [20]. Antigen-antibody complexes were detected using FITC-conjugated anti-mouse IgG. As markers for mucus-secreting pit and neck cells, TRITC-conjugated UEA-1 lectin and FITC-conjugated Grifforia simplifolica II lectin (GSII) were used, respectively. Secondary antibodies and lectins were purchased from Sigma.

Morphometric Studies
Some sections of the mouse stomachs were stained with PAS and hematoxylin and used for estimation of the surface area of different cell types by using an image analysis program (Synoptics Image Analysis, London, UK). For each section, at least three longitudinally cut pits continuous with their glands were chosen for measurements. First, the whole pit-gland unit was outlined and its area was measured in the section and expressed in square micrometers. Then, the areas of pit, parietal, and zymogenic cells were determined. Because in these paraffin sections it was sometimes difficult to differentiate between immature progenitor cells of the isthmus and mucus-secreting neck cells, both were measured together as one category referred to as isthmal-neck cells. The percentage of the area of each cell type in the pit-gland unit was determined, and data were expressed as mean ± SE. Measurements of control versus retinoic acid–treated mice were compared by the Student’s t test.

Terminal Deoxynucleotidyltransferase-Mediated, dUTP Nick-End Labeling Assay
To detect apoptotic cells in control and retinoic acid–treated mice, some sections were processed for terminal deoxynucleotidyltransferase labeling according to the method of Gavrieli et al. [21]. Degoxigenin-labeled UTP was used for the in situ terminal transferase reaction, and incorporation was detected using peroxidase-conjugated anti-digoxigenin and diaminobenzidine (Oncor, MD). Sections were counterstained with PAS.

Electron Microscopy
In some experiments, small pieces of the oxyntic mucosa of control and retinoic acid–treated mice were immersed, immediately after dissection, in 0.1 M sodium cacodylate buffer containing 2% paraformaldehyde and 2.5% gluter-aldehyde. After post-fixation in osmium tetroxide, tissues were dehydrated and processed for resin embedding and ultrathin sections as previously described [1]. Tissue sections were stained with uranyl-lead and examined with Philips electron microscope.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retinoic Acid Enhances BrdU Labeling in the Gastric Glands
When a single injection of BrdU was given to control and retinoic acid–treated mice and stomach sections were immunostained using anti-BrdU antibody, S-phase cells were labeled and found to be localized in the upper portion of the gastric glands (Figs. 1A, 1BGo). When comparing probed sections of control and retinoic acid–treated mice, more S-phase cells were found in the latter. Counts conducted in the gastric glands (n = 16 per mouse) of three different pairs of control and retinoic acid–treated mice revealed that the average number of S-phase cells per gland was approximately two cells in the control mice versus four cells in the retinoic acid–treated mice. In each pair of mice, the difference was statistically significant (p ≤ .001). These dividing cells were found mainly in the isthmus region, with a small proportion in the low pit segment and the neck region (Fig. 2Go, left panel).



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  		Figure 1. One hour (A, B), 1-day (C, D), and 3-day (E, F) BrdU labeling of S-phase cells and their progeny in the oxyntic mucosae of control (A, C, E) and retinoic acid–treated (B, D, F) mice. Sections were incubated with polyclonal anti-BrdU and then with rabbit anti-goat IgG conjugated with peroxidase. Sites of antigen-antibody binding (S-phase nuclei of dividing cells or their progeny) were visualized with diaminobenzidine and appear brown. Sections were counter-stained with periodic acid Schiff to visualize the mucus of pit cells. Note that in the control tissue, 1 hour of BrdU injection results in a few labeled nuclei scattered in a region corresponding to the isthmus. BrdU infusion for 1 and 3 days (C, E) produces an increase in the number of labeled cells that extend upward to the luminal surface and downward to the neck region. In the retinoic acid–treated tissues (B, D, F), BrdU-labeled cells are more numerous and occupy a wider region than in the controls. Labeled cells are seen along the surface and free in the gastric lumen (F). They also expand deep into the basal region. Bar = 140 3m (AD) and 70 3m (E, F). Abbreviations: BrdU, 5-bromo-2'-deoxyuridine; IgG, immunoglobulin G.

 


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  		Figure 2. Percentages of BrdU-labeled cells in three gland regions (isthmus, neck, and base) and two pit segments (high and low) of control (white bars) and retinoic acid–treated (black bars) mice that received BrdU as a single BrdU injection 1 hour before euthanasia (left panel) and as continuous infusion for 1 and 3 days (middle and right panels). Note that BrdU labeling starts mainly in the isthmus and then gradually increases with time and makes its appearance in upper and lower levels. Also note that retinoic acid treatment enhances BrdU labeling in the isthmus and the migration of labeled cells toward the high pit segment and the base region. Abbreviation: BrdU, 5-bromo-2'-deoxyuridine.

 
The increase in dividing progenitors was not restricted to the glandular epithelium of the stomach. The basal progenitor cells of the stratified nonglandular epithelium of the fundus region were also affected by retinoicacid. Counts in three pairs of control and retinoic acid–treated mice revealed that whereas 9% of the basal cells of control fundus were BrdU labeled, this labeling index was significantly increased up to 36% in retinoic acid–treated mice (Figs. 3A, 3BGo).



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  		Figure 3. Labeling of S-phase basal cells of the fundus (A, B) pit and enteroendocrine cells (C, D), neck and zymogenic cells of the oxyntic mucosa (E, F), and mucous and G cells of the pyloric antrum (G, H) of normal (A, C, E, G) and retinoic acid–treated (B, D, F, H) mice. (A, B): Nuclei of S-phase cells appear brown and become more numerous after retinoic acid treatment. (C, D): TRITC-conjugated UEA-1 lectin was used as a marker of pit cells (red), and anti-ghrelin was used to label the cytoplasm of enteroendocrine cells (green). Note that the labeling and distribution of both cell types appear more or less similar in the retinoic acid–treated and control mucosae. (E, F): fluorescein isothiocyanate–conjugated Grifforia simplifolica II lectin was used as a marker of neck cells (green), and anti-intrinsic factor was used to label the cytoplasm of zymogenic cells (red). Note that more zymogenic cells are present in the retinoic acid–treated mucosa. (G, H): TRITC-conjugated UEA1 lectin labels luminal mucous and some mucus-secreting cells (red), and anti-gastrin antibodies label the enteroendocrine G cells (green). Note that no change is seen in the G cell labeling after retinoicacid infusion. Bar=303m (A, B), 90 µm (C, D), and 60 µm (EH). Abbreviations: TRITC, tetramethylrhodamine isothiocyanate; UEA-1, Ulex europaeus agglutinin type 1 lectin.

 
Labeling of all S-phase cells and their progeny in the oxyntic glands during the 1- and 3-day periods of BrdU infusion and comparing it with 1-hour labeling showed that, after their production, cells migrate upward to the luminal surface and downward to the gland bottom (Figs. 1C–1FGo). The area occupied by BrdU-labeled cells was wider in the retinoic acid–treated mice compared with the control (Fig. 1C versus 1DGo and Fig. 1E versus 1FGo). The mucosae of retinoic acid–treated mice showed many labeled cells in the pit segments and along the luminal surface, and some were even seen free in the stomach lumen (Fig. 1FGo).

To visualize and confirm the difference in cell production, BrdU labeling was estimated in the isthmus, neck, and base regions, in addition to the pit segments. Counts of the 1-day BrdU-infused control mice showed an increase in the percentage of labeled cells in the isthmus and the low pit segment and neck region. In addition, very few labeled cells made their appearance in the high pit segment and the base region. Labeled cells were more numerous in retinoic acid–treated mice compared with control ones (Fig. 2Go, middle panel). By 3-day BrdU infusion, counts revealed a further increase in the number of labeled cells, which was more enhanced in the high pit segment and neck and base regions of the retinoic acid–treated mice (Fig. 2Go, right panel).

Retinoic Acid Expands the Area of Zymogenic Cells
Probing of normal and retinoic acid–treated tissue sections with cell markers specific for pit, neck, parietal, and enteroendocrine cells demonstrated no significant difference in the labeled area of these cell types. However, when anti-intrinsic factor was used to probe normal and treated tissues, an increase in the area of the zymogenic cells after retinoic acid treatment was observed (Figs. 3C–3FGo). Measurements of the areas of different cell types confirmed these observations and showed a significant increase in the area occupied by zymogenic cells in the retinoic acid–treated tissues (Fig. 4Go). Zymogenic cell counts in PAS/hematoxylin-stained sections, obtained from three pairs of control and retinoic acid–treated mice, showed that the average number of zymogenic cells in control mice was approximately 11 cells per gland. In retinoic acid–treated mice, the number of zymogenic cells was significantly increased to up to 17 cells per gland.



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  		Figure 4. Measurements of the area of the cell types in the gastric glands of normal (white bars) and retinoic acid–treated (black bars) mice. The percentage of the area of pit, zymogenic, parietal, and isthmal-neck cells are expressed as mean ± standard error of the mean. *Statistically significant difference (p < .0004) between the values of zymogenic cells.

 
Retinoic Acid Enhances the Degeneration of Pit Cells
Because BrdU-labeled pit cells were frequently observed free in the gastric lumen of retinoic acid–treated mice (Fig. 1FGo), it was necessary to confirm this observation and to check for the viability of pit cells at the luminal surface of control versus retinoic acid–treated mice.

The TUNEL assay was used to detect apoptotic cells in the gastric mucosae of control and retinoic acid–treated mice. As previously reported, pit cells normally undergo degeneration at the luminal surface of the gastric mucosa [17]. However, more pit cells at the luminal surface of retinoic acid–treated mice underwent apoptosis compared with control mice (Fig. 5Go). Counts conducted in oxyntic mucosal sections (n = four per mouse) revealed that the number of apoptotic cells was approximately one cell per five glands, whereas in retinoic acid–treated mice, it was approximately three cells per five glands.



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  		Figure 5. Labeling of apoptotic cells in the gastric mucosa of control (A) and retinoic acid–treated (B) mice. Sections were processed for the TUNEL assay as mentioned in the Material and Methods section and then counterstained with periodic acid Schiff. (A, B): Nuclei of apoptotic cells appear brownish (arrows) and are found at the luminal surface (mucus-secreting pit cells). Note that apoptotic cells are more numerous in (B) compared with (A). Bar = 50 µm.

 
Electron microscopic examination of the oxyntic mucosae of normal and retinoic acid–treated mice confirmed the presence of few degenerated cells on the luminal surface of control stomach. In the retinoic acid–treated mice, the degenerated pit cells were numerous. These cells were located along the luminal surface of the mucosa and were characterized by accumulation of vacuolar spaces in the cytoplasm, dissolution of the nucleoplasm, and condensation of the chromatin (Fig. 6Go). Therefore, it seems that retinoic acid enhances the production of pit cells in the isthmus region and low pit segment and enhances their degeneration at the luminal surface.



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  		Figure 6. Electron micrographs showing pit cells of normal (A) and retinoic acid–treated (B) mice as seen at the luminal surface of the oxyntic mucosa. Part of the stomach lumen is seen at the upper right corner, and part of the gland lumen (L) is seen at the left. (A): Pit cells appear elongated, and their cytoplasm contain dark mucous granules. Part of a degenerated pit cell with vacuolated cytoplasm is seen at the bottom (asterisk). (B): Only a few pit cells appear like the normal ones in (A), and six pit cells appear degenerated (asterisks). They have vacuolated cytoplasm, and their nuclei exhibit condensed chromatin. Bar = 5 µm.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study demonstrates that, in mice, 3-day treatment of retinoic acid induces an increase in the proliferation of gastric epithelial progenitors, an increase in pit cell degeneration, and augmentation of zymogenic cell area.

Effects of Retinoic Acid on the Proliferation of Gastric Epithelial Progenitors
Stomach tissues probed with anti-BrdU antibodies showed that the cells that entered the S-phase of the cell cycle are located in the progenitor cell zone of the gland, which confirms earlier observation using 3H-thymidine radioautography [2]. In the present study, more BrdU-labeled cells were present in the retinoic acid–treated tissues compared with the control tissues (Figs. 1A, 1BGo; Fig. 2Go, left panel; Figs. 3A, 3BGo).

To test whether changing the mode of retinoic acid administration would influence its effect on gastric epithelial progenitors, five mice received multiple retinoic acid injections (5 mg/kg body weight per 8 hours) for 3 consecutive days. The S-phase progenitor cells in these retinoic acid–treated mice and their control littermates were labeled by a single injection of BrdU 1 hour before euthanasia. The oxyntic and fundic mucosae of these mice showed an apparent increase in BrdU-labeled progenitor cells after retinoic acid injections, a pattern similar to that of continuous infusion experiment (Figs. 1A, 1BGo; 3A, 3BGo).

To follow the progeny of dividing cells in the oxyntic epithelium, mice received BrdU continuously for 1 or 3 days. All cells that entered the S-phase of the cell cycle during these infusion days incorporated and maintained the BrdU label. Therefore, the overall BrdU labeling in the gastric gland would reflect cell production pattern during this 1- or 3-day infusion period.

In control mice, the comparison of 1-hour with 1-day labeling showed that the continuous infusion of BrdU for 1 day is associated with an increase in the labeled cells in the isthmus region, low pit segment, and neck region (Fig. 2Go, left and middle panels). In addition, some labeled cells make their appearance in the high pit segment and the base region, suggesting cell migration toward the luminal surface and gland bottom. By 3 days, more labeled cells appear in the isthmus and other gland regions (Fig. 2Go, right panel).

Retinoic acid–treated mice showed similar pattern of BrdU labeling compared with control ones. However, retinoic acid stimulates the production of more cells, which migrate and reach the high pit segment and the base region after 1 or 3 day of infusion, compared with control mice (Figs. 1Go, 2Go).

Effects of Retinoic Acid on the Dynamics of Pit Cell Lineage
The present BrdU data support previous observation with 3H-thymidine radioautography that members of the pit cell lineage are produced in the progenitor cell zone and reach the surface epithelium within a few days, where they finally degenerate and are shed [17]. In the retinoic acid–treated mice, there was a stimulation of pit cell production, which rapidly reached the luminal surface. We did not observe any significant change in the area of pit cells after retinoic acid treatment (Fig. 4Go). Also, the average number of pit cells was more or less similar in control and retinoic acid–treated mice (13.1 versus 13.7 cells per pit, respectively). Therefore, it is assumed that there was a steady state in pit cell population. In support of this assumption is the presence of many degenerated and exfoliated pit cells at the luminal surface (Figs. 1FGo, 5BGo, 6BGo).

Recent studies have suggested that degeneration of pit cells depends on interplay between positive regulators of apoptosis such as proapoptotic caspases and negative regulators such as nuclear factor {kappa}B, transforming growth factor ß1, and Bcl-2 [22–24]. Expressions of these factors are known to be regulated by retinoic acid, which has been shown to induce apoptosis in gastric epithelial cells both in vitro and in vivo [25, 26]. Hence, the enhancement of pit cell apoptosis observed in the present study is likely to be attributable to alteration of these factors and provides a further support for the possible role of retinoids in gastric cancer prevention.

To estimate the change in the turnover rate of pit cells after retinoic acid treatment, first, their overall labeling indices were calculated in control and retinoic acid–treated mice 1 hour after injection of BrdU and after its infusion for 1 and 3 days (Table 1Go). Then the labeling indices were plotted against time, and the slope of the regression line, which reflects the increase in cell production rate, was used to calculate pit cell turnover rate (Fig. 7Go). On the assumption that there was a steady state and the rate of cell production equals the rate of cell loss, and from the equation of the regression line, the turnover rate of pit cells in the control mice was 0.84% per hour or 20.2% per day. Therefore, the pit cell turnover time is approximately 5 days. Similarly, in the retinoic acid–treated mice, the turnover rate was estimated at 1.21% per hour, which is 29% per day, and the turnover time was 3.4 days (Fig. 7Go). Therefore, retinoic acid has increased the turnover rate of pit cells and shortened their turnover time.


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  		Table 1. Labeling index (± standard error of the mean) of pit cells of control and retinoic acid–treated mice after 1-hour-injection and 1- and 3-day infusions of BrdU
 


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  		Figure 7. The overall labeling index of pit cells 1 hour after injection of BrdU and after 1 and 3 days of continuous infusion in control and retinoic acid–treated mice. On the assumption that BrdU will remain available in the mouse tissues for 1 hour after its injection to be incorporated by S-phase cells, the values of 1-hour labeling were incorporated in this plot. The slope of the regression line represents the turnover rate, which is 0.84% and 1.16% per hour for control and retinoic acid–treated mice, respectively. Abbreviation: BrdU, 5-bromo-2'-deoxyuridine.

 
When the period of retinoic acid infusion was increased up to 6 days, the overall 3-day BrdU labeling of pit cells did not show significant difference compared with the values of the 3-day treatment (Table 1Go). An increase of BrdU-labeling of pit cells was expected. However, this was not the case, and we have no solid explanation for this observation. It is known that all-trans retinoic acid binds with high affinity to retinoic acid receptors but only very weakly to retinoid X receptors [27]. Perhaps the continuous administration of retinoic acid has an effect on the binding affinity or the turnover of these receptors.

Effects of Retinoic Acid on the Differentiation Program of Zymogenic Cell Lineage
The zymogeniccell lineage includes pre-neck, neck, and zymogenic cells [28]. The present data suggested an enhancement in the production of zymogenic cells in the retinoic acid–treated mice (Fig. 1Go). The stimulation of progenitor cell proliferation was associated with an increase in the area of zymogenic cells (Figs. 3Go, 4Go). These results were not unexpected, because zymogenic cells are long-lived cells (~ 200 days) [28].

In mice, the acid-secreting parietal cells undergo production and maturation in the isthmus followed by a bidirectional mode of migration toward the gland bottom and the luminal surface. The overall turnover time of parietal cells is longer than 50 days [29]. Therefore, as proposed for pit cells, retinoic acid treatment might have accelerated the dynamics of parietal cell turnover by increasing rates of their production and elimination. However, by using the TUNEL assay and electron microscopy, we did not observe any change in their degeneration process. Also, parietal cells maintained a normal steady state without an apparent change in their area compared with the change observed in the immunostaining pattern of zymogenic cells, the longest-lived cells in the epithelium. So, it seems that an effect of retinoic acid on the dynamics of parietal cells of 1- to 2-month old mice is unlikely.

Whether the effects of retinoic acid on the gastric epithelial cell dynamics are attributable to direct binding to their receptors or other factors remain to be investigated. We tested the possibility that the effects observed are part of a general trophic effect, possibly by an increase in the production of gastrin-secreting G cells in the pyloric antrum. However, immunoprobing of these cells in control and retinoic acid–treated mice did not reveal any significant difference (Figs. 3C, 3DGo). Insulin could be another factor, because we have recently found it to stimulate proliferation of gastric epithelial progenitors in vitro [30] and retinoic acid has been reported to stimulate insulin production [31]. Finally, because we have observed effects for retinoic acid on epithelial progenitors and two lineages, pit and zymogenic, migrating in different directions and the mRNA and protein expressions of retinoid receptors are localized in more than one cell type in the gastric glands [12, 13], it seems likely that the effects observed in this study due to retinoic acid treatment are via ligand receptor binding.

Recently, the expression of retinoic acid–synthesizing enzyme, retinaldehyde dehydrogenase 1 (RALDH1), has been demonstrated in the proliferating/differentiating gastric epithelium of developing mice [32]. Because cell proliferation and differentiation also occur in adult stomach, and because the present study demonstrates that retinoic acid alters the dynamics of the gastric epithelium of adult mice, it seems very likely that RALDH1 expression is maintained in the adult mouse stomach and retinoic acid synthesis contributes to autocrine control of gastric epithelial homeostasis.

In conclusion, our results indicate that retinoic acid plays a role in controlling cell proliferation and influencing decisions of cell commitment program in the gastric glands of the mouse stomach. These data suggest that retinoic acid is needed to maintain normal cell proliferation and differentiation in the gastric glands, which may provide a possible explanation for its cytoprotective effects against gastric mucosal ulceration and tumor development.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to thank Dr. Gerald Buzzell for his critical comments on the manuscript. This work was carried out with the support of grant NP/02/22 from the Faculty of Medicine and Health Sciences, United Arab Emirates University, to S.M.K.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received August 5, 2004; accepted for publication November 18, 2004.




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