Expression of Reg family genes in the gastrointestinal tract of mice treated with indomethacin
Abstract
Regenerating gene (Reg) family proteins, which are classified into four types, commonly act as trophic and/or antiapoptotic factors in gastrointestinal (GI) diseases. However, it remains unclear how these proteins coordinate their similar roles under such pathophysiological conditions. Here, we investigated the interrelationships of Reg family gene expression with mucosal cell proliferation and apoptosis in nonsteroidal anti-inflammatory drug (NSAID)-induced GI injury. GI injury was induced by subcutaneous injection of indomethacin into Reg I knockout (KO) and wild-type (WT) mice, and its severity was scored histopathologically. Temporal changes in the expression of Reg family genes, mucosal proliferation, and apoptosis were evaluated throughout the GI tract by real-time RT-PCR, Ki-67 immunoreactivity, and TUNEL assay, respectively. Reg I, Reg III family, and Reg IV were predominantly expressed in the upper, middle, and lower GI mucosa, respectively. Expression of Reg I and Reg III family genes was upregulated in specific portions of the GI tract after indomethacin treatment. Ki-67-positive epithelial cells were significantly decreased in the gastric and small-intestinal mucosa of Reg I KO mice under normal conditions. After treatment with indomethacin, the number of TUNEL-positive cells was significantly greater throughout the GI mucosa in Reg I KO mice than in WT mice. Expression of Reg I was independent of that of other Reg family genes in, not only normal GI tissues, but also indomethacin-induced GI lesions. Members of the Reg gene family show distinct profiles of expression in the GI tract, and Reg I independently plays a role in protecting the GI mucosa against NSAID-induced injury.
nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed worldwide for patients with arthritis or cardiac and cerebrovascular diseases (16). Upper gastrointestinal (GI) damage is a major adverse effect of NSAID use (16), and, moreover, recent evidence suggests that NSAIDs are responsible for mucosal injury, not only in the upper, but also the lower GI tract (10, 16). Prostaglandin synthesis is considered to play a pivotal role in the mechanism of NSAID-induced GI damage (21); however, its pathophysiology still remains to be elucidated.
The regenerating gene (Reg) was originally isolated from rat regenerating pancreatic islet cells, and its human homolog was named REG Iα (23). Recently, many Reg-related genes have been isolated and shown to constitute a multigene family (types I–IV) (8). We have previously reported that REG Iα and REG IV are involved in the pathophysiology of GI inflammation (5, 17) and that, moreover, they act as trophic and/or antiapoptotic factors under inflammatory conditions (17, 18). Other investigators have reported that Reg III family genes are overexpressed in inflamed GI mucosa (11, 13) and that their products, including REG Iα and REG IV proteins, may exert trophic and/or antiapoptotic effects on pancreatic, hepatic, or GI epithelial cells (2, 12, 14). On the other hand, these findings raise questions as to why these proteins with similar functions are produced in the GI mucosa and what kind of interrelationships exist among the genes of this family. In the present study, therefore, we first investigated the profiles of expression of Reg family genes in the GI tract. We then used Reg I knockout (KO) mice to examine the role of Reg I in NSAID-induced mucosal injury in the GI tract, focusing on the relationship between the expression of Reg I and that of other genes in the Reg family.
MATERIALS AND METHODS
Animal model.
Reg I KO (Reg I−/−) mice and wild-type (WT) (Reg I+/+) littermates were used for the following experiments. The Reg I KO mice were generated on an ICR background as previously described (24). All mice were maintained in cages on a 12-h:12-h light/dark cycle under specific pathogen-free conditions.
Mice received subcutaneous indomethacin (Sigma, St. Louis, MO; 20 mg/kg in 5% NaHCO3) and were killed in a time-dependent manner. The GI tissues were removed from mice, cut open along the longitudinal axis, rinsed with saline, and fixed in neutral aqueous phosphate-buffered 10% formalin for histological examinations or stored in nitrogen liquid for real-time RT-PCR. Regarding the stomach tissues, we excluded the forestomach from the obtained samples but did not separate the remaining samples into the antrum and body parts. This animal experiment was carried out with the approval of the Animal Use and Care Committee at Hyogo College of Medicine.
Histological evaluation.
When we removed the GI tissues, the length of the small intestine and the colon was measured. The tissues fixed as above were embedded in paraffin, cut perpendicularly to the surface at 4-μm thickness, and stained by hematoxylin and eosin. To evaluate the damage of GI tissues, we used a previously proposed histological damage-scoring system (6), as shown in Table 1.
| Histological Factors | Score |
|---|---|
| Width of ulceration | |
| No ulcer | 0 |
| Small ulcer, <3 mm | 1 |
| Large ulcer, >3 mm | 2 |
| Depth of lesion | |
| None | 0 |
| Mucosa | 1 |
| Submucosa | 2 |
| Muscularis propria | 3 |
| Serosa | 4 |
| Inflammation | |
| None | 0 |
| Mild | 1 |
| Moderate | 2 |
| Severe | 3 |
| Thrombi | |
| No | 0 |
| Yes | 1 |
Real-time RT-PCR.
Total RNA was isolated from GI tissues with Trizol reagent (Invitrogen, Carlsbad, CA). Total RNA (4 μg) was reverse-transcribed using oligo-dT primer (Applied Biosystems, Branchburg, NJ), and real-time RT-PCR was performed using 7900H Fast Real-Time PCR System (Applied Biosystems), as previously described (19). The set of primers for mouse Reg I, Reg II, Reg IIIα, Reg IIIβ, Reg IIIγ, Reg IIIδ, Reg IV, and GAPDH were prepared as shown in Table 2. Real-time RT-PCR assays were carried out with 200 ng of RNA equivalent cDNA, SYBR Green Master Mix (Applied Biosystems), and 500 nmol/l gene-specific primers. The PCR cycling conditions were 50°C for 15 s and 60°C for 60 s. The intensity of the fluorescent dye was determined, and the expression levels of Reg family mRNA were normalized to GAPDH mRNA expression levels. The normalized value <10−3 was determined as 0.
| Gene | Direction | Primer Sequence |
|---|---|---|
| Reg I | Forward | 5′-GAACGCCTACTTCATCCTGC-3′ |
| Reverse | 5′-GATGGCAGGTCTTCTTCAGC-3′ | |
| Reg II | Forward | 5′-GATCAGCATGGCTCAGAACA-3′ |
| Reverse | 5′-TCTTCAGCTACCTGGCCTTG-3′ | |
| Reg IIIα | Forward | 5′-CTCAGGACATCTCGTGTCTATTCTT-3′ |
| Reverse | 5′-AGTGACCACGGTTGACAGTAGAG-3′ | |
| Reg IIIβ | Forward | 5′-TCCCAGGCTTATGGCTCCTA-3′ |
| Reverse | 5′-GCAGGCCAGTTCTGCATCA-3′ | |
| Reg IIIγ | Forward | 5′-TTCCTGTCCTCCATGATCAAAA-3′ |
| Reverse | 5′-CATCCACCTCTGTTGGGTTCA-3′ | |
| Reg IIIδ | Forward | 5′-TGGAACCACAGACCTGGGCTA-3′ |
| Reverse | 5′-GAGCAGAAATGCCAGGTGTCC-3′ | |
| Reg IV | Forward | 5′-CGCTGAGATGAACCCCAAG-3′ |
| Reverse | 5′-TGAGAGGGAAGTGGGAAGAG-3′ | |
| GAPDH | Forward | 5′-GGAGAAACCTGCCAAGTATG-3′ |
| Reverse | 5′-TGGGAGTTGCTGTTGAAGTC-3′ |
Immunohistochemistry.
Immunohistochemical stainings for Ki-67 and Reg I protein were performed with an Envision Kit (Dako, Kyoto, Japan) as previously described (1), using anti-mouse Ki-67 antibody (clone TEC-3; Dako; dilution 1:200) and anti-rat Reg I antibody (dilution 1:2,000). The tissue sections of the GI tracts were also used for TUNEL assay using an in situ cell death detection kit (Roche, Mannheim, Germany), as previously described (5). In brief, the sections were deparaffinized, rehydrated, and then incubated with proteinase K (20 μg/ml) for 15 min at 37°C. The sections were washed in PBS, and endogenous peroxidase was blocked by 3% H2O2 in methanol for 30 min at room temperature. After being washed in PBS, the sections were incubated with terminal transferase buffer and reaction mixture for 60 min at 37°C. The slides were then washed in PBS and incubated with peroxidase-conjugated Fab fragments of anti-fluorescein at 37°C for 30 min. The slides were washed in PBS, visualized by 3,3′-diaminobenzide tetrahydrochloride with 0.05% H2O2 for 3 min, and then counterstained with Mayer's hematoxylin. To evaluate cell proliferation and apoptosis, 20 well-oriented gastric glands, small intestinal crypt-villi, and colonic crypts were scored in sections from each of the mice. The indices for cell proliferation and apoptosis were expressed as the number of Ki-67- and TUNEL-positive epithelial cells per gland/crypt-villi/crypt, respectively.
Statistical analysis.
All values were expressed as the means ± SE. Significance of differences between two animal groups was analyzed by Mann-Whitney U-test. The change of gene expression level in two genotype groups was compared by two-factor factorial ANOVA. Differences were considered to be significant at P < 0.05.
RESULTS
Expression profile of Reg family genes in the normal gastrointestinal tract.
Under normal conditions in WT mice (Fig. 1A), Reg I expression was predominant from the stomach to the ileum, and its level peaked from the duodenum to the jejunum. Reg I expression was also detected in the colon at a low level. Expression of Reg II was hardly detectable throughout the GI tract. Reg III family genes such as Reg IIIα, Reg IIIβ, and Reg IIIγ were expressed mainly from the duodenum to the ileum and at a low level in the colon. Reg IIIδ was expressed at a low level throughout the GI tract. Reg IV expression was predominant from the duodenum to the distal colon, and its level peaked in the colon. None of the Reg family genes were detectable in the esophagus (data not shown).
Fig. 1.Expression profile of Reg family genes in the gastrointestinal tract in wild-type (WT) (A) and Reg I knockout (KO) (B) mice. Results are expressed as the means ± SE (n = 4). S, stomach; D, duodenum; J, jejunum; I, ileum; PC, proximal colon; DC, distal colon.
We also examined the expression profile of Reg family genes throughout the GI tract in Reg I KO mice (Fig. 1B). As a matter of course, Reg I expression was never detected throughout the GI tract. Interestingly, for other Reg family genes, the patterns of change in their expression levels were similar in both WT and Reg I KO mice, suggesting that loss of Reg I may not affect the expression profile of other Reg family genes in the GI tract under normal conditions.
Changes in macroscopic and microscopic histology in the gastrointestinal tract of mice treated with indomethacin.
Some of the Reg I KO mice (3 of 15; 20%) died during this experiment when they received indomethacin at the dose of 20 mg/kg, whereas no WT mice died after receiving this dosage (Fig. 2A). At 72 h after indomethacin treatment, the small intestine was significantly shorter in Reg I KO mice than that in WT mice (Fig. 2B), and, similarly, the colon was also significantly shorter in the former than in the latter (Fig. 2B). Figure 2C shows the macroscopic histology of GI tract tissues from the experimental mice treated with indomethacin. In WT mice, visible lesions were hardly evident macroscopically under these experimental conditions. However, in Reg I KO mice, gastric ulcers were observed in the antrum, erosions were evident in the small-intestinal mucosa, and faint redness was present in the colonic mucosa (Fig. 2C). Consistent with the macroscopic findings, microscopic examination revealed large gastric ulcers and severe mucosal damage in the small intestine in Reg I KO mice treated with indomethacin (Fig. 3). On the other hand, the mucosal injury in WT mice was apparently weaker than that in Reg I KO mice. In WT mice, Reg I was strongly expressed in the antral and small-intestinal mucosa after treatment with indomethacin (Fig. 3).
Fig. 2.Reg I deficiency renders mice susceptible to indomethacin-induced mucosal injury in the gastrointestinal tract. A: survival rate of WT and Reg I KO mice treated with indomethacin. B: length of small intestine and colon in WT and Reg I KO mice treated with indomethacin. C: photographs showing representative macroscopic findings of the gastrointestinal mucosa in WT and Reg I KO mice treated with indomethacin. Arrows indicate a gastric ulcer lesion. Bottom: magnification of outlined areas in middle. Results are expressed as the means ± SE (n = 4) at each time point. The samples from dead mice were not included in analyses. *P < 0.05 vs. WT group at the same time point.
Fig. 3.Microscopic findings of the gastrointestinal mucosa in WT and Reg I KO mice treated with indomethacin. In WT mice with indomethacin treatment, Reg I was strongly expressed in glandular cells in the antrum. In their small intestine, Reg I was strongly expressed from the villi to the upper crypt cells. HE, hematoxylin and eosin staining; Reg I, immunostaining of Reg I.
Using microscopy, we then assessed the histological damage in experimental mice (Fig. 4). The histological damage scores for Reg I KO mice were significantly higher than those for WT mice throughout the GI tract at 24 and 72 h after indomethacin treatment. In Reg I KO mice, the histological damage tended to be highest in the stomach, followed in order by the small intestine and colon. This pattern was similar in WT mice, but the damage was moderate (<5) in the stomach and weak (<3) in the small intestine and colon.
Fig. 4.Change in histological damage score in the gastrointestinal tract in WT and Reg I KO mice treated with indomethacin. Results are expressed as the means ± SE (n = 4). *P < 0.05; **P < 0.01 vs. WT group at the same time point.
Changes in expression of Reg family genes in the GI tract of mice treated with indomethacin.
In the stomach of WT mice, expression of Reg I, Reg IIIβ, Reg IIIγ, and Reg IIIδ was significantly increased after indomethacin treatment. In the stomach of Reg I KO mice, expression of Reg I was abolished, but that of Reg IIIβ, Reg IIIγ, and Reg IIIδ was significantly increased, as was the case in WT mice. Reg II expression was additionally increased in the stomach of Reg I KO mice after indomethacin-treatment although its maximum level was not very high (Fig. 5).
Fig. 5.Changes in expression of Reg family genes in the gastrointestinal tract in WT and Reg I KO mice treated with indomethacin. ●, Reg I; ×, Reg II; ■, Reg IIIα; ▲, Reg IIIβ; □, Reg IIIγ; △, Reg IIIδ; ○, Reg IV. Results are expressed as the means ± SE (n = 4). *Significantly increased at either time point vs. 0 h. The gene whose expression level is significantly different between WT and Reg I KO mice was marked by square.
In the duodenum and jejunum of WT mice, expression of Reg I, Reg IIIβ, and Reg IIIγ was significantly increased after indomethacin treatment. In the same tissues of Reg I KO mice, Reg I expression was abolished, but Reg IIIβ and Reg IIIγ expression was significantly increased, as was the case in WT mice. Reg II and Reg IIIδ expression was additionally increased at a low level in the duodenum and jejunum of indomethacin-treated Reg I KO mice, respectively. On the other hand, in the ileum, none of the Reg II, Reg III family, or Reg IV showed a significant change of expression in either WT or Reg I KO mice (Fig. 5).
In the proximal colon of WT mice, expression of Reg IIIα, Reg IIIβ, and Reg IIIγ was significantly increased after indomethacin treatment, whereas no change was evident in other gene expression. A similar pattern was observed in Reg I KO mice. In the distal colon of WT mice, expression of Reg IIIα, Reg IIIβ, and Reg IIIδ was significantly increased after indomethacin treatment, whereas other genes expression remained unchanged. This pattern was the same as that in Reg I KO mice (Fig. 5).
In the comparison of the gene expression level between Reg I KO and WT mice, Reg I expression was greater throughout the GI tract in WT than in Reg I KO mice. Reg II and Reg IV expression was additionally increased in the gastroduodenum and colon of indomethacin-treated Reg I KO mice, respectively. Reg IIIβ and Reg IIIδ expression in the stomach was different between indomethacin-treated WT and Reg I KO mice. In addition, Reg IIIγ expression in the distal colon was different between indomethacin-treated WT and Reg I KO mice (Fig. 5).
Effects of cell proliferation and apoptosis by indomethacin with and without Reg I.
Reg I KO mice had fewer basal proliferative cells in the antrum (Fig. 6A), and, unlike the WT, the number did not increase significantly in response to indomethacin treatment (Fig. 6B). Similarly, Reg I KO mice had fewer proliferative cells in the small intestinal crypts. The number of Ki-67-positive cells was not affected in the small intestine of Reg I KO or WT mice under this dose of indomethacin treatment. In the colonic mucosa, no significant difference was found between WT and Reg I KO mice, either with or without indomethacin treatment.
Fig. 6.A: immunostaining of Ki-67 in the gastrointestinal mucosa of WT and Reg I KO mice treated with indomethacin (IND). B: scores for Ki-67-positive cells in the gastrointestinal mucosa of the experimental mice. Results are expressed as the means ± SE (n = 4). Cont, no treatment; IND, 24 h after indomethacin treatment (20 mg/kg sc). Significant difference by ANOVA: *P < 0.05; **P < 0.01.
TUNEL-positive cells were observed throughout the GI mucosa of both WT and Reg I KO mice after indomethacin administration (Fig. 7A). Under normal conditions, there were no differences between WT and Reg I KO mice in the number of TUNEL-positive cells throughout the GI mucosa. Indomethacin treatment significantly increased the number of TUNEL-positive epithelial cells from the stomach to the small intestine in WT mice or throughout the GI tract in Reg I KO mice (Fig. 7B). When mice were treated with indomethacin, the number of TUNEL-positive cells was significantly greater throughout the GI mucosa of Reg I KO mice than that of WT mice.
Fig. 7.A: TUNEL detection of apoptotic cells in the gastrointestinal mucosa of WT and Reg I KO mice treated with indomethacin. Representative photographs show gastrointestinal mucosa of mice at 24 h after treatment with indomethacin (20 mg/kg sc). B: scores for TUNEL-positive cells in the gastrointestinal mucosa of the experimental mice. Results are expressed as the means ± SE (n = 4). Significant difference by ANOVA: *P < 0.05; **P < 0.01.
DISCUSSION
It has been accepted that REG family proteins commonly act as trophic and/or antiapoptotic factors following GI inflammation and its associated cancer development (2, 12, 14, 17, 18). Moreover, several microarray analyses have isolated Reg family genes as novel molecules overexpressed in inflammatory bowel disease (3, 11), colorectal cancers (27), the gastritis-gastric cancer sequence (7, 20), and pancreatic cancers (15), and, therefore, much attention has been focused on the roles of REG family proteins in the GI tract. In the present study, to clarify why similar family proteins are concomitantly produced in the GI mucosa, we first investigated the expression profiles of Reg family genes throughout the GI tract. Interestingly, as shown in Fig. 1, Reg I, Reg III, and Reg IV expression was observed predominantly in the upper GI tract, small intestine, and lower GI tract, respectively, suggesting that Reg family genes may share their responsible portions in the GI tract. Furthermore, we investigated the relationship between Reg I and other Reg family genes by comparing the profile of their expression in the GI tract of Reg I-deficient mice with that of WT mice. No remarkable differences were observed in the expression of Reg II, Reg III family, and Reg IV expression between Reg I-deficient and WT mice although we had expected supplementary expression of non-Reg I genes in Reg I-deficient mice. This finding suggests that Reg I is independent of other Reg family genes.
In the present study, we also examined the role of Reg I and its association with other Reg family genes in NSAID-induced GI injury. NSAID treatment caused mucosal injury in the upper GI tract of WT mice under the experimental conditions employed. Subsequently, GI mucosal injury was apparently more severe in Reg I-deficient than in WT mice, compatible with a previous report (6). This suggests that loss of Reg I is susceptible to NSAID-induced GI damage, whereas, on the other hand, we wondered whether other Reg family genes would compensate for the lost role of Reg I. As shown in Fig. 5, not only Reg I, but also Reg IIIβ and Reg IIIγ were significantly upregulated in the upper GI during the process of NSAID-induced mucosal injury. In Reg I-deficient mice, the patterns of change in non-Reg I gene expression were very similar to those in WT mice although Reg II and Reg IV were additionally upregulated at a low level in the gastroduodenum and colon, respectively. These finding suggest that Reg I is independent of other Reg family genes in, not only normal GI tissues, but also NSAID-induced GI lesions. Conversely, the difference in severity of NSAID-induced GI lesions between WT and Reg I-deficient mice was clearly associated with the loss of Reg I gene expression. Besides, we observed that NSAID treatment induced mucosal injury in the lower as well as the upper GI tract of Reg I-deficient mice although the tissue damage was not so severe in the lower GI tract. Reg III family genes are likely to act predominantly in the lower GI tract, and, therefore, we cannot support confidently the involvement of Reg I gene in NSAID-induced lower GI lesions. However, because REG family proteins are secreted into the GI tract lumen (26, 27), it may be interesting to speculate that REG I protein produced in the upper GI tract may act in the lower GI tract as well.
The effects of REG family protein on epithelial cell proliferation and antiapoptosis may be important for protecting the GI mucosa from NSAID-induced injury. Therefore, we investigated cell proliferation and apoptosis histologically in experimental mice treated with NSAID. With regard to cell proliferation, it was noteworthy that epithelial cell proliferation was significantly suppressed in the upper GI tract of Reg I-deficient mice without treatment. This finding suggests that Reg I plays a pivotal role in the maintenance of the upper GI mucosa in terms of cell proliferation. When GI mucosal tissue was injured, epithelial cell proliferation was normally promoted to heal the injured epithelium (22). This behavior was consistently observed in the gastric mucosa of WT mice with NSAID treatment, whereas it was obvious in the small intestine and colon because of weak tissue damage in WT mice. Analysis of Reg I-deficient mice with NSAID treatment suggested that epithelial cell proliferation tended to be promoted in GI mucosa. This finding may be reasonable because, not only Reg I, but also various growth factors play roles in cell proliferation in the healing process of injured GI mucosa (22). On the other hand, we confirmed that epithelial cell apoptosis is significantly enhanced throughout GI mucosa in Reg I-deficient mice with NSAID treatment although other Reg family genes are upregulated as in WT mice with same treatment. Therefore, it is tempting to speculate that Reg I is important in determining resistance to epithelial cell apoptosis associated with NSAID-induced GI damage.
In summary, we have shown that the Reg family shows a predominant portion for their gene expression in the GI tract, respectively, and that Reg I and Reg III family gene expression is upregulated in their predominant GI portion of WT with NSAID treatment. Furthermore, the present study suggested that Reg I is independent of other Reg family genes in NSAID-induced lesions as well as normal tissues in the GI tract and that Reg I plays a role in GI mucosal integrity by promoting cell proliferation and conferring resistance to apoptotic stimuli. REG family proteins belong to the calcium-dependent lectin (C-type lectin) superfamily (4) and have recently been shown to act, not only as trophic and/or antiapoptotic factors (2, 12, 14, 17, 18), but also as antimicrobial peptides (9, 25). These effects may be of consistent benefit for protection of the GI mucosa from chemical or infectious injury, and, therefore, investigators have started intensive studies of REG family proteins in the GI tract. In this context, the present study has obtained comprehensive information regarding expression of Reg family genes in NSAID-induced GI mucosal injury and may give some idea to future studies.
GRANTS
This work was supported in part by Grants-in-Aid for Scientific Research
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: C.S., H.F., K.H., Y.K., H.E., M.Y., H.Y., T.T., T.O., J.W., and S.T. performed experiments; C.S., H.F., K.H., and H.Y. analyzed data; C.S., H.F., H.Y., T.O., J.W., and S.T. interpreted results of experiments; C.S. and H.F. prepared figures; C.S. and H.F. drafted manuscript; C.S., H.F., S.T., T.C., and H.M. edited and revised manuscript; C.S., H.F., K.H., Y.K., H.E., M.Y., H.Y., T.T., T.O., J.W., S.T., T.C., and H.M. approved final version of manuscript; H.F. conception and design of research.
ACKNOWLEDGMENTS
We thank Noriko Kamiya and Mayumi Yamada (Hyogo College of Medicine) for technical assistance.
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