Kex2 family endoprotease furin is expressed specifically in pit-region parietal cells of the rat gastric mucosa

Hitoshi Kamimura1,2, Yoshitaka Konda1, Hiromi Yokota1, Sei-Ichi Takenoshita2, Yukio Nagamachi2, Hiroyuki Kuwano2, and Toshiyuki Takeuchi1

1 Department of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma University, and 2 First Department of Surgery, Gunma University School of Medicine, Maebashi 371, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The proprotein-processing endoprotease furin is localized in the gastric epithelial cells of the pit region in the rat gastric gland. The gastric pit is composed of several cell types, including gastric surface mucosal (GSM) cells and parietal cells. Furin converts many growth- or differentiation-related proproteins to their active forms. We examined identification of furin-positive cells by immunostaining of rat gastric mucosa and regulators of the furin expression by measuring the furin promoter activity by luciferase assay. Furin-positive cells were stained for H+-K+-ATPase, indicating that they are parietal cells. Furin-positive parietal cells were not stained for transforming growth factor-alpha (TGF-alpha ) but were surrounded by TGF-alpha -positive GSM cells. In contrast, parietal cells below the proliferative zone were positive for TGF-alpha but not for furin. Furin-positive parietal cells expressed a high level of epidermal growth factor receptor (EGFR). TGF-alpha stimulated the furin promoter activity highly in a mouse GSM cell line GSM06. Thus we suggest that the parietal cells of the pit region have furin-mediated functions that can be stimulated by EGFR signaling.

transforming growth factor-alpha ; gastric surface mucous cells; gastric pit


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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THE GASTRIC PIT REGION is composed of several cell types, including gastric surface mucous (GSM) cells (also called pit cells) and parietal cells (17-19). These cells arise from granule-free precursor cells in the proliferative zone at the isthmus of the gastric gland. From the proliferative zone, GSM cells move upward to the luminal surface of gastric glands, with a lifetime of ~3 days in mice (19). Parietal cells move both upward and downward; the ratio of upward-moving and downward-moving cells was reported as half and half (17). In the pit, parietal cells distribute from the middle of the pit to near the proliferative zone and have a lifetime of 54 days in mice. The parietal cells develop mostly from the parietal cell precursors to preparietal cells, but a fraction of parietal cells develop from prepit cell precursors and preneck cell precursors (17). Thus prepit cell precursors have the potential to develop into either prepit or preparietal cells.

GSM cells develop into their well-differentiated form in three stages: a prepit cell stage, a mature pit cell stage, and a final top-pit cell stage. Cell-stage differentiation is well characterized by the appearance of mucus-containing secretory granules that begin to appear at the prepit cell stage and increase in great number to the mature pit cell stage (15, 19). In addition to these mucus-containing granules, the cells express transforming growth factor-alpha (TGF-alpha ) with differentiation (13, 29). TGF-alpha shares a common receptor with epidermal growth factor (EGF), EGFR, and with heparin-binding EGF-like growth factor (HB-EGF) (26). EGFR is expressed in parietal cells and GSM cells (30, 34). Thus TGF-alpha and EGFR are abundantly expressed in the pit region. Although TGF-alpha is known to induce cell proliferation in primary-cultured rat GSM cells (5), its receptor distribution in terminally differentiated GSM and parietal cells may suggest that TGF-alpha exerts nonproliferative functions in the gastric gland.

Recently, we found that the proprotein-processing endoprotease furin is distributed around the upper one-fourth of the gastric gland of the adult rat (21). This furin-positive cell zone is a little higher than the proliferative zone. Furin cleaves the carboxy-terminal end of -Arg-4-X-(Lys/Arg)-2-Arg-1, which is found in many growth-related precursor proteins (14, 28). These precursor proteins include adhesion molecules such as a procadherin family and integrin subunits alpha 3 and alpha 6, matrix metalloproteinases (MMP) such as stromelysin-3 and membrane-type MMP, several growth factor precursors including platelet-derived growth factor, human HB-EGF (27), transforming growth factor-beta , and parathyroid hormone-related protein, and, in some growth factor proreceptors such as insulin receptor, insulin-like growth factor-1 receptor and hepatocyte growth factor receptor (oncoprotein MET). Because many of these precursor proteins with furin-cleavable sites are expressed in the gastric mucosa, furin may be involved in the growth and differentiation of mucosal cells.

We previously investigated the expression of furin and TGF-alpha using a GSM cell line, GSM06 (21). This cell line is derived from the GSM cells of a transgenic mouse that was transformed with the temperature-sensitive SV40 T antigen (35). At high temperature (39°C), at which the SV40 T antigen is inactivated, the GSM06 cells exhibit growth arrest and exhibit differentiated features, such as the production of periodic acid-Schiff-positive materials, secretory granules, and expression of TGF-alpha . In contrast, when the cells are exposed to 33°C, they start growing by acquiring a characteristic of T antigen-transformed cells and express a high level of furin and a diminished low level of TGF-alpha .

In the rat gastric pit, furin-positive cells are localized a little higher than the proliferative zone (21). Because furin-positive GSM06 cells become TGF-alpha positive when placed in a differentiation-inducing condition (39°C and/or confluent state), we presumed that furin-positive cells may become TGF-alpha -positive cells during the pit cell lineage differentiation in the normal gastric mucosa. In this paper, we investigated the topological relation of furin-positive and TGF-alpha -positive cells in the rat gastric glands, the cellular characteristics of the furin-positive cells, and the effect of TGF-alpha as well as other growth factors on the expression of furin.


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Morphological studies. Male Wistar rats (150-200 g) were purchased from Imai Experimental Animal Farm (Saitama, Japan). The rats were maintained under controlled light (7:00 AM to 7:00 PM) with food and water provided ad libitum. When their stomachs were used for morphological studies, they were fasted for 24 h and then killed. Stomachs were minced and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C for 24 h. Small pieces of minced tissue underwent saccharose replacement and were embedded in OCT compound for frozen sectioning by a microtome.

The antibody to mouse furin was prepared in rabbits as described previously (21). Monoclonal antibody to H+-K+-ATPase was obtained by injecting rabbit parietal cell-microsomal fractions into mice. We obtained antibody to proliferative cell nuclear antigen (PCNA) from DAKO (Glostrup, Denmark), monoclonal antibody to TGF-alpha from Calbiochem-Novabiochem (San Diego, CA), and sheep antibody to EGFR from Upstate Biotechnology (Lake Placid, NY). F-actin was stained with FITC-labeled phalloidin (Molecular Probes, San Diego, CA).

Tissue sections were probed with the above-described antibody to furin, PCNA, TGF-alpha , EGFR, or H+-K+-ATPase as a first antibody. They were then incubated with either indodicarbocyanine-conjugated affinity-purified donkey anti-rabbit or anti-sheep IgG (Jackson ImmunoResearch, West Grove, PA) or FITC-labeled anti-rabbit IgG (Jackson ImmunoResearch) as a secondary antibody. The secondary antibody IgG was selected depending on the species on which the first antibody was raised. For horseradish peroxidase (HRP)-catalyzing staining, an LSAB2/HRP staining kit (DAKO) was used as the secondary antibody reaction system. The HRP reaction was visualized with 0.1% H2O2 and 3-amino-9-ethylcarbazole (brown) or 3,3'-diaminobenzidine hydrochloride (black). The specimens were examined with an Olympus BX50 microscope with an incident illuminator.

Stimulation of acid secretion. Physiological saline (100 µl) containing 100 µM histamine and carbachol (300 mg/kg body wt) was injected subcutaneously into rats. After 30 min, the stomachs were removed and subjected to the above-described fixation and immunostaining process.

Plasmid construction. A human furin 5'-upstream sequence was cloned from the human genomic library using oligonucleotide probes based on the previously reported sequence (2). The furin gene possesses the three alternative 5'-noncoding exons. Of the three, only exon 1 has a TATA box and is activated by transcription factor CAAT enhancer binding protein-beta (C/EBP-beta ). The other two, exons 1A and 1B, have characteristics of promoters for housekeeping genes. Thus we used the TATA box containing furin promoter in this study. Four 5'-upstream DNA fragments, -3633/+55 (Pst I-Pst I), -1092/+55 (Bgl II-Pst I), -612/+55 (Xba I-Pst I), and -56/+55 (BamH I-Pst I), were placed before a firefly luciferase gene supplied with the Dual-Luciferase reporter assay system (Promega, Madison, WI).

Cell culture and luciferase assay. As a culture model for the surface mucosal cells, we used a GSM06 cell line (35). The cells were cultured on a collagen type I-coated plastic plate (Iwaki, Tokyo, Japan) in DMEM-Ham's F-12 medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (GIBCO, Grand Island, NY) and 1% ITES (100% ITES: 2 mg/l insulin, 2 mg/l transferrin, 0.122 mg/l ethanolamine, and 9.14 µg/l sodium selenite) in a humidified 5% CO2 atmosphere at 33°C unless otherwise indicated. Luciferase activity was assayed by transient gene expression in GSM06 cells. GSM06 cells were seeded initially at 5 × 105 cells/10-cm plate and cultured for 2 days. Transfection was carried out with each of the five luciferase gene constructs, including a promoterless gene, together with the sea pansy luciferase gene placed under several ubiquitous promoters as an internal standard, using a DOSPER liposomal transfection reagent (Boehringer Manheim, Mannheim, Germany). Twenty-four hours after the transfection, the medium was changed to a new one and the culture was continued for another 6 h. The cells were then harvested to prepare cell lysates using the cell lysis buffer supplied with the Dual-Luciferase reporter assay system (Promega). Stimulants were obtained from the following suppliers: phorbol 12-myristate 13-acetate (PMA) from Sigma Chemical, TGF-alpha and EGF from Collaborative Biomedical Products (Bedford, MA), and platelet-derived growth factor-BB from Genzyme (Cambridge, MA). Mitogen-activated protein (MAP) kinase kinase (MEK) inhibitor PD-098059 was obtained from Research Biochemicals International (Natick, MA). Protein kinase C (PKC) inhibitor H-7 was obtained from Seikagaku (Tokyo, Japan). Each of these stimulants was added at the change of medium, 24 h after transfection; the cells were then incubated for another 6 h before harvest. The reaction was initiated by the addition of 100 µl of the luciferin solution to 50 µl of cell lysate (~100 µg protein), and light emission was measured for 10 s using a luminometer. Luciferase activity was measured as arbitrary light units per 10 s per 100 µg protein. Protein concentrations were determined by the Bradford method. Luciferase activity is expressed as multiplicity (-fold) against the value obtained by the promoterless luciferase gene or as a percentage against the control value obtained without stimulants.


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Distribution of furin-positive cells in the rat gastric mucosa. We examined the distribution of PCNA-positive cells, furin-positive cells, and TGF-alpha -positive cells in the rat gastric mucosa (Fig. 1, A-F). Black staining of PCNA was localized to nuclei in the gastric mucosal cells, which have a relatively small cell size (Fig. 1, D and E). Most PCNA-positive cells were distributed around one-third down the gastric gland relative to the luminal surface, although there were several positive cells in the glandular region (Fig. 1, A and B). Overlapping with a higher level of the PCNA-positive cell layer, the furin-positive cells were distributed upward in a scatter pattern around one-fourth down the gastric gland relative to the luminal surface (brown cells in Fig. 1A and black cells in Fig. 1C). Furin-positive cells looked larger than PCNA-positive cells (Fig. 1, D and F). In Fig. 1E, large TGF-alpha -negative cells appeared to be furin-positive when compared with furin-positive cells in Fig. 1, D and F. The furin-positive cells decreased in number toward the luminal surface, but staining became heavier and occupied the entire cytoplasm (Fig. 1, D and F). Furin-positive staining was also observed in the lower glandular cells, whose distribution may be due to chief cells, as determined by their location (Fig. 1, A and C). TGF-alpha -positive cells were distributed further upward in the pit region, but the luminal top-pit region was negative for TGF-alpha (Fig. 1, B and C). TGF-alpha staining, apparently in the parietal cells of the glandular region, was distinctly weaker than that of GSM cells in the pit (Fig. 1, B and C). Furin-positive but TGF-alpha -negative cells were surrounded by TGF-alpha -positive GSM cells (Fig. 1, E and F). Thus furin-positive cells were scattered between the PCNA-positive and TGF-alpha -positive cell layers in the pit region.


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Fig. 1.   Immunostaining of rat gastric mucosa for proliferative cell nuclear antigen (PCNA), furin, and transforming growth factor-alpha (TGF-alpha ). Immunostainings were visualized with horseradish peroxidase reaction. A-C are ×200. D-F are ×400. A and D: furin (brown) and PCNA (black). B and E: TGF-alpha (brown) and PCNA (black). C and F: TGF-alpha (brown) and furin (black).

Immunohistochemical characterization of furin-positive cells. Because furin-positive cells looked larger in size and because parietal cells were distributed in the pit region as well as the glandular region, we suspected that the furin-positive cells in the gastric pit might be parietal cells. To examine this possibility, we performed double immunostaining of furin and H+-K+-ATPase because H+-K+-ATPase is specifically localized to the tubulovesicles of the resting parietal cells. The H+-K+-ATPase-positive parietal cells distributed from the pit region to the glandular region of the gastric gland (Fig. 2A). As expected, furin-positive cells were also positive for H+-K+-ATPase, although H+-K+-ATPase-positive cells were not always positive for furin in the pit region (Fig. 2). The population ratio of furin-positive parietal cells to the total parietal cells located up from the middle of the growth zone was 30.1 ± 2.8% (n = 8). The staining of H+-K+-ATPase in the pit-region parietal cells indicated a complex tubulovesicle-canaliculus system composed of many small curly formations (Fig. 2D). Furin staining distributed along with H+-K+-ATPase staining in these cells (Fig. 2, D-F).


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Fig. 2.   Immunostainings of rat gastric mucosa for H+-K+-ATPase and furin. A-C are ×200. D-F are ×1,000. A and D: H+-K+-ATPase staining by FITC. B and E: furin staining by indodicarbocyanine (Cy3). C and F: double staining.

Because tubulovesicles fuse with intracellular canaliculi when the parietal cells have been stimulated with acid secretagogues, we stimulated acid secretion with histamine and carbachol. After stimulation, both H+-K+-ATPase and furin were overlapped together and displayed a horseshoe-shape formation (Fig. 3). Thus, by light microscopic resolution, furin appears to be colocalized with H+-K+-ATPase in the pit-region parietal cells.


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Fig. 3.   Immunostainings of rat gastric mucosa for H+-K+-ATPase and furin after stimulation with histamine and carbachol (×1,000). Thirty minutes after the subcutaneous injection of histamine (0.1 mM/0.1 ml) and carbachol (300 µg/kg body wt), gastric mucosa was taken for immunostaining. A: H+-K+-ATPase staining by FITC. B: furin staining by Cy3. C: double staining.

Furin-positive parietal cells stain positively for H+-K+-ATPase in a curly formation. We compared the appearance of parietal cells over and below the proliferative zone using H+-K+-ATPase and phalloidin stainings because a tubulovesicle-canaliculus system is associated with actin filament (F-actin) cytoskeletons in parietal cells and FITC-phalloidin specifically stains actin filaments (19). Phalloidin staining as well as H+-K+-ATPase staining were spread to the entire cytoplasm in the pit-region parietal cells (Fig. 4, A and B). In contrast, in the parietal cells of the glandular region, H+-K+-ATPase distributed with many punctate stainings around the nucleus (Fig. 4E). Actin filament cytoskeletons surrounded the H+-K+-ATPase-positive circular formation with a partial overlap inside (Fig. 4F). Thus parietal cells can be classified into two subtypes: the furin-positive but TGF-alpha -negative cell population that is typically found in the pit region and the TGF-alpha -positive but furin-negative cell population in the glandular region, with the former looking larger than the latter.


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Fig. 4.   Staining of rat gastric mucosa for F-actin and H+-K+-ATPase. A-C show mucosal cells in the pit region (×1,000). D-F show mucosal cells in the glandular region (×1,000). A and D: F-actin staining with FITC-phalloidin. B and E: H+-K+-ATPase staining with Cy3. C and F: double staining.

EGF receptor localization in the pit region. Because furin-positive parietal cells were surrounded by TGF-alpha -positive GSM cells in the pit region (Fig. 1, C and F) and because TGF-alpha is situated extracellularly on its membrane-bound precursor (24), we examined the distribution of its receptor EGFR in the gastric pit. EGFR was stained over the whole gastric mucosa, especially in the top-pit and glandular regions (Fig. 5B), as reported previously (30, 34). In the pit, parietal cells were stained strongly for EGFR (Fig. 5, D and E). The H+-K+-ATPase-containing tubulovesicle-canaliculus system looked surrounded by EGFR-positive stainings (Fig. 5F). GSM cells were apparently stained for EGFR in a punctate manner but much weaker than the staining of parietal cells (Fig. 5, D and E). In contrast, the parietal cells of the glandular region were stained for EGFR less strongly than the pit-region parietal cells (Fig. 5, G and H). Instead, we found strong EGFR-positive cells distributed next to parietal cells (Fig. 5C and arrows in Fig. 5, G-I). By their distribution pattern, these EGFR-positive cells appeared to be chief cells. In general, EGFR-positive parietal cells are surrounded by TGF-alpha -positive GSM cells in the gastric pit, whereas TGF-alpha -positive parietal cells were neighbored by strongly EGFR-positive cells in the glandular region.


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Fig. 5.   Immunostaining of rat gastric mucosa for H+-K+-ATPase and epidermal growth factor receptor (EGFR). A-C are ×100. D-F show mucosal cells in the pit region (×1,000). G-I show mucosal cells in the glandular region (×1,000). A, D, and G: H+-K+-ATPase staining with FITC. B, E, and H: EGFR staining with Cy3. C, F, and I: double staining. Arrows in G-I, EGFR-positive cells distributed next to parietal cells.

Effect of TGF-alpha and other stimulants on the expression of furin. GSM cells turn over with a lifetime of ~3 days, whereas parietal cells possess a lifetime of more than 50 days (17, 19). Thus, during the movement of TGF-alpha -positive GSM cells upward among EGFR-positive and furin-positive parietal cells in the gastric pit, the EGFR-positive parietal cells may receive juxtacrine signals from TGF-alpha -positive GSM cells by cell-to-cell interaction. To examine this possibility, we measured furin expression by TGF-alpha and other stimulants with a furin promoter plus luciferase reporter gene. Although the parietal cell is the best choice as a target to investigate furin promoter activity, it is hard to isolate and introduce DNA into parietal cells. To circumvent this difficulty, we used a GSM06 cell line that expresses EGFR (data not shown).

Initially, we characterized a promoter activity of furin gene by using five lengths of 5'-upstream sequence and found that the luciferase activity was highest for the -612/+55-length promoter (Fig. 6A). The fact that the two longer furin promoters presented lower luciferase activity suggests the presence of inhibitory elements between -1092 and -612. Furthermore, the rapid decrease of luciferase activity by the -56/+55 promoter may suggest the presence of enhancing elements between -612 and -55. We used the -612/+55-length promoter for the following study. Among five stimulants, including TGF-alpha , EGF, insulin-like growth factor I, hepatocyte growth factor, and PMA, TGF-alpha and PMA were highest and EGF was significantly higher in inducing luciferase activity (Fig. 6B). Furthermore, luciferase activity was increased dose dependently by both TGF-alpha and PMA up to 10 nM, then plateaued to a 100 nM point (Fig. 6C). It is suggested that furin promoter activity is regulated by the EGFR-mediated signaling and PKC-mediated signaling, probably through a Ras/MAP kinase signaling pathway (9, 23). We examined the signaling through the Ras/MAP kinase pathway by using the MEK inhibitor PD-098059 (8) and confirmed that the increase of furin promoter activity by TGF-alpha was suppressed by this inhibitor in a dose-dependent manner (Fig. 6D). The furin promoter activity without TGF-alpha also decreased gradually, suggesting that the Ras/MAP kinase pathway is activated to some extent in GSM06 cells, possibly by endogenously produced TGF-alpha (21). We further examined the functional role of PKC on furin expression by a PKC inhibitor H-7 (11) and PKC desensitization by PMA. As shown in Fig. 6E, in the presence of H-7, luciferase activity decreased 25.0% even in nonstimulated GSM06 cells, suggesting the production of endogenous stimulants including TGF-alpha from GSM06 cells. The activity by TGF-alpha and PMA stimulation decreased 48.3% and 68.4%, respectively, in the presence of H-7 (Fig. 6E). After pretreatment of the cells with 1 µM PMA for 24 h, TGF-alpha increased the luciferase activity only 12.6% relative to the value without PMA (data not shown). Thus TGF-alpha signal appears to be mediated partly by PKC. Recently, Polk (32) showed that PMA pretreatment of mouse small intestine epithelium-derived culture cells blocked EGF-induced cell migration and demonstrated that phospholipase C is involved in EGF signaling by producing diacylglycerol for PKC activation in a Ca2+-dependent manner.


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Fig. 6.   Furin promoter activity assay by TGF-alpha and other stimulants. A: activity of furin promoter; 5'-flanking region of the human furin gene was deleted by restriction endonucleases and fused to the firefly luciferase cDNA. DNA constructs were transfected to the gastric surface mucosal cell line, GSM06, and luciferase activity was measured 12 h later. Data are presented as multiplicity (-fold) of increase against the promoterless luciferase DNA construct. Values represent means ± SD. B: furin promoter activity by TGF-alpha and other stimulants; 5'-flanking region of the human furin promoter (-612/+55) was utilized for this experiment. Stimulants used were TGF-alpha , EGF, insulin-like growth factor I (IGF-I), hepatocyte growth factor (HGF), and phorbol 12-myristate 13-acetate (PMA) at concentrations of 10 nM each. Data are represented as -fold against the promoter activity without stimulants. Values represent means ± SD. * P < 0.05, ** P < 0.01 against the control value. C: dose-response curves of luciferase activity by TGF-alpha and PMA. Luciferase activity without stimulation was taken as 100. Values represent means ± SD. ** P < 0.01 against the control value. D: suppression of luciferase activity by mitogen-activated protein kinase kinase inhibitor PD-098059. Luciferase activity was assayed in the presence or absence of 10 nM TGF-alpha together with an increasing dose of PD-098059 (0, 10, 30, and 100 µM). Luciferase activity without PD-098059 in the absence of TGF-alpha was taken as 100. open circle , In the presence of TGF-alpha . , In the absence of TGF-alpha . Values represent means ± SD. E: suppression of luciferase activity by protein kinase C inhibitor H-7. Luciferase activity was assayed in the presence or absence of 10 µM H-7. Luciferase activity without H-7 in the absence of stimulants was taken as 100. Stimulants used were TGF-alpha (10 ng/ml) and PMA (100 nM). Open bars, in the absence of H-7. Solid bars, in the presence of H-7. Values represent means ± SD.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The present study demonstrates that furin-positive cells localized in the pit region were identified as parietal cells by the presence of H+-K+-ATPase. The furin-positive parietal cells were not stained for TGF-alpha , in contrast to the parietal cells in the glandular region. Instead, the furin-positive parietal cells were surrounded by TGF-alpha -positive GSM cells, suggesting signaling from TGF-alpha -positive GSM cells to EGFR-positive parietal cells in the pit region. We further demonstrated that furin expression was upregulated by TGF-alpha using a GSM cell-derived cell line, GSM06.

Most parietal cells are derived from the granule-free precursor cells localized in the isthmus, from where GSM cells are also derived (17). By morphological appearance, parietal cells are apparently similar in any region, except those in the lower base. Karam et al. (20) reported functional heterogeneity of parietal cells along the pit-gland axis by showing very little morphological change in response to acid secretagogues, in contrast to the typical morphological transformation of parietal cells in other regions. In this study, we demonstrate that parietal cells displayed different features over and below the proliferative zone in the isthmus. The distinct features of parietal cells in the pit region were their larger size, the complex form of the tubulovesicle-canaliculus system, expression of furin, and absence of TGF-alpha , compared with those in the neck to base regions.

Parietal cells that possess EGFR stay in the pit for 50-60 days, whereas TGF-alpha -positive GSM cells migrate upward from the proliferative zone to the top-pit region by 3 days (17, 19). Thus, when GSM cells pass by parietal cells, both cell types may exchange important signals for cell differentiation by cell-to-cell interaction. Using canine oxyntic mucosal cell culture, Chen et al. (4) demonstrated that GSM cells that are in contact with parietal cells display DNA synthesis and suggested that TGF-alpha from parietal cells stimulates the growth of GSM cells in vivo. However, in the rat gastric pit, parietal cells do not express TGF-alpha but express EGFR extensively, whereas GSM cells express TGF-alpha strongly and EGFR weakly. Thus we think that the TGF-alpha signal moves from GSM cells to parietal cells in the pit. In the glandular region, parietal cells express TGF-alpha to a moderate extent, so TGF-alpha -positive parietal cells can stimulate EGFR-positive GSM cells in the mixed culture of gastric mucosal cells (4).

The absence of PCNA-positive staining suggests that parietal cells and GSM cells were not proliferating and thus that TGF-alpha -to-EGFR signaling in the pit may not be growth promoting but rather cell differentiating. Recently, Miyoshi et al. (25) demonstrated that the membrane-bound form of HB-EGF suppresses cell growth and promotes survival of rat hepatoma-derived AH66tc cells; in contrast, a soluble form of HB-EGF cleaved from the membrane-bound form increases cell growth of AH66tc cells. Takemura et al. (36) also showed a distinct feature of soluble and membrane-bound forms of HB-EGF using a renal epithelial cell line, NRK-52E. HB-EGF as well as TGF-alpha precursors are thought to be processed to their active forms by MMP-like endoproteases (12). Thus the effect of membrane-bound TGF-alpha on GSM cell membranes may maintain the survival of EGFR-expressing parietal cells, resulting in a long lifespan for parietal cells.

TGF-alpha is known to suppress acid secretion acutely from parietal cells (33, 38), but its chronic effect increases acid secretion in isolated parietal cells (6). We suspected that parietal cells in the pit display similar acid secretion function to those in the glandular region. The parietal cells displayed horseshoe-shaped morphological transformation when stimulated with acid secretagogues. Thus we think that acid secretion is possible in the pit-region parietal cells under strong TGF-alpha signaling. Interestingly, Kaise et al. (16) demonstrated that EGF increases the promoter activity of H+-K+-ATPase alpha -subunit gene in isolated canine parietal cells (16). We also measured a furin promoter activity using luciferase as a reporter gene and found that TGF-alpha and the phorbol ester PMA stimulate furin gene expression in GSM06 cells. Although caution is required when extrapolating this result to in vivo gastric mucosa, we presume that TGF-alpha signaling from GSM cell membranes stimulates furin gene expression as well as H+-K+-ATPase alpha -subunit gene expression in parietal cells (16).

TGF-alpha is released from its membrane-bound precursor by metalloproteinases (1), which are stimulated by PMA through a PKC-mediated pathway (10, 31). It is important to identify the natural stimulants leading to metalloproteinase activation through this PKC-mediated pathway in the gastric mucosa. Although furin does not activate TGF-alpha processing and also TGF-alpha -activating metalloproteinase processing, furin expressed by EGFR signaling may activate other growth-related proteins to their active forms that facilitate cell growth or differentiation in an autocrine and/or paracrine fashion. Several candidate proproteins can be nominated, including procadherin, integrin subunits alpha 3 and alpha 6, membrane-type MMP MT1-MMP, and parathyroid hormone-related protein, as described in the introduction (14, 28). The pit-region parietal cells may produce furin-activated proteins and/or secrete a truncated form of furin itself that activates proproteins in extracellular space (37). After activation by furin, these proteins may exert their effect on GSM cells. At present, these substrate proteins remain to be identified.

Parietal cells play a pivotal role in maintaining gastric mucosal cell structure because parietal cell-deleted mouse mucosa exhibit atrophic gastritis-like appearance (3, 22). In this study, we found that parietal cells are distinct in the expression of furin and TGF-alpha over and below the proliferative zone. We suggest that pit-region parietal cells play an essential role in gastric pit structure formation by producing furin-mediated growth and differentiation factors in contact with TGF-alpha -positive GSM cells.


    ACKNOWLEDGEMENTS

We thank Dr. Norifumi Sugiyama, Molecular Research Laboratory, Daiichi Pharmaceutical, Tokyo 134, for providing a GSM06 cell line; Dr. Kentaro Sugano, Health Care Center, University of Tokyo, and Dr. Kazuhisa Nakayama, Gene Experiment Center, Tsukuba University, for helpful discussions; and Reiko Uchida for secretarial assistance.


    FOOTNOTES

This work is supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: T. Takeuchi, Dept. of Molecular Medicine, Institute for Molecular and Cellular Regulation, Gunma Univ., Showa-machi, Maebashi 371-8512, Japan (E-mail: tstake{at}news.sb.gunma-u.ac.jp).

Received 11 November 1998; accepted in final form 11 April 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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