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Altered expression of a putative progenitor cell marker DCAMKL1 in the rat gastric mucosa in regeneration, metaplasia and dysplasia
© Kikuchi et al; licensee BioMed Central Ltd. 2010
Received: 9 February 2010
Accepted: 18 June 2010
Published: 18 June 2010
Doublecortin and calcium/calmodulin-dependent protein kinase-like-1 (DCAMKL1) is a candidate marker for progenitor cells in the gastrointestinal mucosa. Lineage cells in the gastric mucosa are derived from progenitor cells, but this process can be altered after injury. Therefore, we explored DCAMKL1 expression under pathological conditions.
An immunohistochemical analysis was performed in rat stomach with acute superficial injury, chronic ulcer, intestinal metaplasia and dysplasia.
DCAMKL1 was exclusively expressed in immature quiescent cells in the isthmus of normal fundic glands, where putative progenitor cells are thought to reside. DCAMKL1-positive cells and proliferating cells shed into the lumen after superficial injury and re-appeared during the regenerative process, mainly in the superficial mucosa. In the marginal mucosa around the active ulcer, parietal and chief cells diminished, foveolar hyperplasia was evident, and trefoil factor family 2 (TFF2)/spasmolytic polypeptide-expressing metaplasia (SPEM) emerged at the gland base. DCAMKL1 cells re-emerged in the deep mucosa juxtaposed with SPEM and proliferating cells. In the healing ulcer, the TFF2 cell population expanded and seemed to redifferentiate to chief cells, while proliferating cells and DCAMKL1 cells appeared above and below the TFF2 cells to promote healing. SPEM appeared and PCNA cells increased in the intestinalized mucosa, and DCAMKL1 was expressed in the proximity of the PCNA cells in the deep mucosa. DCAMKL1, PCNA and TFF2 were expressed in different dysplastic cells lining dilated glands near SPEM.
The ultrastructural appearance of DCAMKL1-positive cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 expression is closely associated with TFF2/SPEM cells after injury. DCAMKL1 cells repopulate close to proliferating, hyperplastic, metaplastic and dysplastic cells, and the progenitor zone shifts according to the pathological circumstances.
The mouse and human gastric unit shows monoclonal conversion, indicating the presence of multipotent stem cells[1, 2]. Electron microscopic autoradiography in mouse have implied that granule-free cells in the isthmus act as multipotent stem cells. The differentiation and migration processes of cell lineages can be altered by injury. The progenitor cell zone in the isthmus is easily damaged by intraluminal ethanol, non-steroidal anti-inflammatory drugs or Helicobacter pylori (H. pylori). Chronic inflammation of the stomach can lead to atrophy and specialized cell loss as tangible effects of tissue-specific progenitor cell injury or loss. However, the behavior of progenitor cells after acute or chronic mucosal damage and the mechanism of restoration of these cells during mucosal regeneration are not well understood.
The progenitor cell population is important in maintenance and regeneration of the gastric epithelium, but long-lived progenitor cells are at risk of accumulating mutations that lead to cancer. Neoplasia can follow cellular metaplasia due to chronic inflammation and repair. However, precise analysis of the role and alteration of progenitor cells in the sequence of gastritis-metaplasia-dysplasia-cancer has not been performed, mainly due to the lack of discrete progenitor cell markers in the stomach.
Musashi-1, a marker of progenitor cells in the mouse small intestine, is not expressed in putative progenitor cells, but is found in parietal cells in the rat fundic isthmus. The villin-1 promoter/enhancer fragment is a marker of possible gastric progenitor cells in the isthmus of the pyloric glands. A lineage study indicated that the intestinal progenitor cell marker Lgr5 is expressed at the base of prospective fundic and pyloric glands in the neonatal stomach, whereas expression in adult was predominantly restricted to the base of the pyloric glands. Thus, there are no definite markers for progenitors in adult fundic glands.
DCAMKL1 is one of the products of Gene Ontogeny-enriched transcripts found in comparison with mouse gastric and small intestinal progenitor datasets. Immunohistochemical analysis using a DCAMKL1 antibody revealed single cell staining in intestinal crypt sections at or near position 4 and in gastric isthmus cells. After the first report, specific localization of DCAMKL1-expressing cells in a stem cell niche was shown in the small intestine of mice[9, 10] and in the colon of mice and humans[11, 12], whereas DCAMKL1 was coexpressed with Musashi-1 in parietal cells in the stomach of mice.
The first aim of this study was to determine whether DCAMKL1 is a marker for progenitor cells in the rat stomach. The second aim was to elucidate the temporal and spatial patterns of appearance of cells specifically expressing DCAMKL1 under several gastric pathological conditions.
All animal protocols were approved by the Keio University Animal Research Committee. Male Wistar rats weighing about 200 g were fasted for 24 hours with free access to water. To produce acute superficial injury in the fundic mucosa, absolute ethanol (1 ml) was instilled into the stomach by gastric intubation. To produce chronic deep ulcers, 20% acetic acid (50 μl) was injected into the fundic submucosa of the anterior wall using a microsyringe. The rats were euthanized 3 days and 1, 2 and 3 weeks after the acetic acid injection.
The method for inducing intestinal metaplasia in the rat gastric mucosa has been described elsewhere[14, 15]. Briefly, rats received two X-ray doses of 10 Gy each and were euthanized 6 months after irradiation. The method for inducing dysplasia in the rat gastric mucosa has also been described elsewhere. Briefly, 50 μg/ml of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Aldrich Chemical Co., Milwaukee, WI) was given to rats ad libitum for 4 months.
Rabbit anti-DCAMKL1 immunoglobulin (Ig)G (Abcam, Cambridge, UK; final dilution 1:100), mouse anti-proliferating cell nuclear antigen (PCNA) IgG (DAKO, Carpinteria, CA; ready to use), rabbit anti-PCNA IgG (Abcam; 1:200), mouse anti-H+/K+-adenosine triphosphatase (ATPase) α subunit IgG (Research Diagnostics Inc. Flanders, NJ; 1:200), sheep anti-pepsinogen II IgG (United States Biological, Swampscott, MA; 1:100), mouse anti-MUC6 IgM (Kanto Kagaku, Tokyo; 1:100), mouse anti-MUC5AC IgG (Abcam; 1:100), mouse anti-TFF2 IgM (Abcam; 1:200), guinea pig anti-histidine decarboxylase (HDC) IgG (ARP, Belmont, MA; 1:100), goat anti-ghrelin IgG (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100), and mouse anti-somatostatin IgG (Biomeda, Foster City, CA; 1:25) were used as primary antibodies.
The stomach tissue was fixed in 10% neutral-buffered formalin overnight, and then embedded in paraffin and sectioned (4 μm). The sections were stained with hematoxylin and eosin (H&E) using standard techniques. Periodic acid-Schiff (PAS)-Alcian Blue staining was performed to detect mucous cell lineages.
For immunohistochemical analysis, paraffin-embedded sections were deparaffinized and pretreated with an appropriate retrieval procedure for each antigen. Sections were incubated in 0.3% H2O2 in methanol for 10 minutes to inactivate endogenous peroxidase and then washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST). After incubation with blocking solution (Block Ace, Dainippon Seiyaku, Tokyo, Japan) for 10 minutes, the sections were incubated with a primary antibody for 1 hour at room temperature. Thereafter, each step was followed by washing 3 times with PBST for 3 minutes. The sections were incubated with HP-conjugated IgG or IgM for 40 minutes. The labeled cells were colored brown with 3,3'-diaminobenzidine hydrochloride (DAB) using a DAB reagent set (DAKO), and then counterstained with Mayer's hematoxylin.
For double-color immunostaining, an indirect immunoalkaline phosphatase method was used following the indirect immunoperoxidase procedure described above. After reaction with DAB, the sections were incubated with a second primary antibody for 1 hour, followed by incubation with ALP-conjugated IgG or IgM for 40 minutes. The labeled cells were stained blue with an ALP substrate kit III (Vector Blue, Vector Laboratories, Burlingame, CA).
For antigen retrieval of pepsinogen II and MUC6, proteinase K (DAKO) was applied topically to the deparaffinized sections for 6 minutes. For antigen retrieval of PCNA (mouse IgG), the sections were heated in distilled water in an autoclave for 10 minutes. For antigen retrieval of MUC5AC, the sections were heated in citrate buffer in an autoclave for 10 minutes. No antigen retrieval procedure was used for DCAMKL1, H+/K+-ATPase, PCNA (rabbit IgG), TFF2, HDC, ghrelin or somatostatin staining.
Scoring of DCAMKL1 Cells and PCNA Cells
Sections that underwent double-color immunostaining using DCAMKL1 and PCNA were analyzed to determine the number of immunostained cells. Sections were used from 5 rats, and 10 well-oriented gastric units were analyzed in each section.
Pre-embedding immunoperoxidase electron microscopy was carried out as follows. Small pieces of fresh specimens from the gastric corpus of untreated rats were fixed in 4% paraformaldehyde for 24 hours, followed by fixation in 0.1% glutaraldehyde and 4% paraformaldehyde for 1 hour. Cryostat sections (6 μm) were prepared and incubated with anti-DCAMKL1 antibody for 48 hours, followed by incubation with HP-conjugated anti-rabbit IgG for 24 hours. The sections were then fixed with 0.5% glutaraldehyde for 5 minutes, reacted with DAB, post-fixed with 2% osmium tetroxide, dehydrated through a graded ethanol series, and embedded in epoxy resin. Ultrathin sections were cut with an ultramicrotome and stained in uranyl acetate solution and lead citrate solution. The specimens were examined using a transmission electron microscope (JEM-1200EX, JEOL, Tokyo, Japan).
Normal Fundic Gland
Acute Superficial Mucosal Injury and Rapid Renewal
We then explored DCAMKL1 expression in the marginal mucosa of the active ulcer. Dispersed DCAMKL1-expressing cells were present close to MUC5AC cell linings (Figure 5i) and juxtaposed with SPEM cells (Figure 5j). PCNA cells were also distributed in the vicinity of SPEM cells and some SPEM cells coexpressed PCNA (Figure 5k), implying that the SPEM lineage is multiplying and proliferative. DCAMKL1 cells were intermingled with PCNA cells at the base of the gland, but did not coexpress PCNA (Figure 5l). This indicates that DCAMKL1 cells are maintained in a quiescent state. The progenitor zone of PCNA cells and DCAMKL1 cells was displaced from the isthmus in the normal gland to the base in the margin of the active ulcer.
Intestinal Metaplasia and Dysplasia
The study showed that DCAMKL1-expressing cells are exclusively present in the isthmus of the rat fundic gland, in which multipotent progenitor cells are thought to reside[1, 3]. We demonstrated that DCAMKL1 cells are discrete from differentiated epithelial cells, including endocrine cell lineages. Furthermore, immunoelectron microscopy showed that DCAMKL1 was expressed in immature epithelial cells with few organelles, which correspond to the granule-free cells previously proposed as presumptive progenitor cells in the gastric isthmus. DCAMKL1 is coexpressed with Musashi-1 in parietal cells in the mouse stomach, whereas this study demonstrated that DCAMKL1 cells were distinct from parietal cells, which express Musashi-1 in the rat stomach.
A recent report has shown that PCNA-positive proliferating cells contain prefoveolar cells. The increased number of proliferating cells are probably derived from progenitor cells, but the process of progenitor cell repopulation after injury is obscure. In this study, DCAMKL1 cells were recruited prior to restoration of proliferating cells, and the number and distribution of DCAMKL1 cells changed almost concomitantly with those of proliferating cells after 24 hours. This finding implies that DCAMKL1-expressing cells are progenitor cells that give rise to proliferating cells. Several DCAMKL1 cells and PCNA cells were present at the mucosal surface during the early regenerative period. Transient displacement of the progenitor zone toward the surface may help to facilitate rapid and preferential restoration of foveolar cells, which is the lineage that is most damaged and lost in superficial mucosal injury. Since the recruited DCAMKL1 cells reappeared within a day after the indigenous cells were lost, the repopulating DCAMKL1 cells in the injured mucosa may migrate from non-injured glands or recruit from progeny of a multipotent stem cell lineage that undergoes continuous expansion or extinction in the niche[9, 22].
Chronic mucosal ulceration in the gastrointestinal tract initiates a healing process from the mucosal base at the ulcer edge, and can induce novel cell lineages corresponding to SPEM. These appear to be derived from multipotent stem cells in the crypt of the small intestine and colon, whereas SPEM and proliferating cells at the base of the margin of the gastric ulcer is distant from the normal progenitor zone in the isthmus. The chronic ulcer model in this study developed several weeks after treatment, which makes migration of bone marrow-derived stem cells unlikely because engraftment of these cells as gastric epithelial cells occurs in mice after sustained H. felis infection over more than 1 year, but not in mice with a gastric ulcer induced by acetic acid. The presence of a second progenitor population, cryptic progenitor cells in the stomach, has been predicted for many years[16, 24], but has yet to be identified. In this study, putative progenitor DCAMKL1 cells were found in the vicinity of two mucous cell lineages, foveolar cells and SPEM cells, in hyperplasia at the ulcer margin. DCAMKL1 cells juxtaposed with SPEM are compatible with the cryptic progenitor cells. The intestinal progenitor cell marker Lgr5 is present at the base of glands and not in the isthmus, and that a progenitor cell population increased markedly during inflammation. We hypothesize that DCAMKL1-expressing cells at the base of the gastric gland are the second-line progenitor population, which is masked under physiological conditions. These dormant progenitors may be activated by inflammatory cytokines during ulcer formation, and may play a pivotal role in initiating the healing process.
DCAMKL1 cells and PCNA cells were present close to TFF2/SPEM cells in the ulcer margin. TFF2 has a healing function in the stomach, but the mechanism is not fully understood. The function of TFF2, which is protease resistant and produces mucus/TFF complexes of high viscosity, might be to protect the progenitor zone . During ulcer healing, the TFF2 cell population expanded from the base to the neck of glands. Unlike mucous neck cells in the normal mucosa, TFF2 cells in the healing ulcer coexpressed MUC5AC (but little MUC6) in the neck and also coexpressed pepsinogen. This suggests that SPEM cells redifferentiate to chief cells in the process of ulcer healing.
Alterations of cell lineages in dysplasia are illustrated in Figure 11B. Cystic dilation of glands was elicited and the morphological appearance was consistent with gastritis cystic profunda as a precursor to dysplasia and neoplasia, which develops in Helicobacter-infected animals and humans[23, 31]. The cystic regions invaded the submucosa and were accompanied by SPEM, which also expanded to the submucosa from the base of the mucosa. It is of note that several studies have suggested that SPEM is a precursor of gastric cancer[23, 27, 32]. DCAMKL1 and TFF2 were expressed in dilated glands in this study. Expression of DCAMKL1 has been reported in mouse adenoma and human colorectal cancer, and thus DCAMKL1 and TFF2 are putative markers for gastric carcinogenesis. A recent report showed that DCAMKL1 is a positive regulator of tumorigenesis in the colon, but the function of DCAMKL1 in the gastrointestinal tract is largely unknown.
The ultrastructural appearance of DCAMKL1 cells and the expression patterns of DCAMKL1 in normal and pathological states indicate that the cells belong to a progenitor cell population. DCAMKL1 shows different expression profiles in ulcer healing, intestinal metaplasia and dysplasia, while DCAMKL1 expression is closely associated with TFF2/SPEM cells.
DCAMKL1 is expressed close to proliferating, hyperplastic, metaplastic and dysplastic cells, and the progenitor zone shifts according to the pathological circumstances.
The authors are extremely grateful to Mr. Hideaki Hasegawa and Mrs. Yoshiko Itoh (Teaching and Research Support Center, Tokai University School of Medicine) for their excellent technical assistance with electron microscopy.
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