ARTICLE |
Correspondence to: Toshihiko Iwanaga, Lab. of Anatomy, Graduate School of Veterinary Medicine, Hokkaido University, Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan. E-mail: tiwanaga@vetmed.hokudai.ac.jp
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Summary |
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Recently, the second mammalian chitinase, designated acidic mammalian chitinase (AMCase), has been identified in human, mouse, and cow. In contrast to the earlier identified macrophage-derived chitinase (chitotriosidase), this chitinase is richly expressed in the gastrointestinal (GI) tract, suggesting its role in digestion of chitin-containing foods as well as defense against chitin-coated microorganisms and parasites. This in situ hybridization study first revealed cellular localization of the gut-type chitinase in the mouse and chicken. In adult mice, the parotid gland, von Ebner's gland, and gastric chief cells, all of which are exocrine cells of the serous type, expressed the gut chitinase mRNA. In the chicken, oxyntico-peptic cells in glandular stomach (proventriculus) and hepatocytes expressed the chitinase mRNA. Because cattle produce the gut chitinase (chitin-binding protein b04) only in the liver, the gut chitinases in mammals and birds have three major sources of production, i.e., the salivary gland, stomach, and liver. During ontogenetic development, the expression level in the parotid gland and stomach of mice increased to the adult level before weaning, whereas in the stomach of chickens intense signals were detectable in embryos from incubation day 7. (J Histochem Cytochem 50:10811089, 2002)
Key Words: chitinase, acidic mammalian chitinase, pepsinogen, in situ hybridization, gastrointestinal tract, parotid gland
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Introduction |
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CHITIN is the most abundant glycopolymer that constitutes the integument of many species, such as insect exoskeletons, shells of crustaceans, and fungal cell walls (
Independently of these studies, our research group (
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Materials and Methods |
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Probes for In Situ Hybridization
Three non-overlapping antisense oligonucleotides were used for in situ hybridization (ISH). They were complementary to nucleotide residues 81125 (tatttcaccaactgggcccagtatcggccaggtctggggagcttc), 541585 (ttactgctgctgtagctggtgggatttccaacatccaggctggct), and 11901234 (tcctgacgtgccttccgagccagtgactactcctccaggaagtgg) of mouse gut chitinase (AMCase) cDNA (
Tissue Samples
Male adult ddY mice, 8 weeks old, were used for the present ISH. The animals were sacrificed by dislocation of cervical vertebrae, and the salivary glands (parotid gland, submandibular gland, and sublingual gland), lips, tongue, palate, stomach, various regions of the intestine (from duodenum to rectum), liver, pancreas, lung, kidney, adrenal gland, and urogenital tract were rapidly removed and frozen in liquid nitrogen. The salivary glands and stomach were also collected from neonatal ddY mice of both sexes at 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 days of age. Cryostat sections about 14 µm thick, were prepared and mounted on glass slides precoated with 3-aminopropyltriethoxysilane (Shinetsu Chemical Industry; Tokyo, Japan).
For chicken samples, the oral mucosa, tongue, crop, proventriculus, gizzard, different regions of the intestine (from duodenum to rectum), liver, lung, pancreas, kidney, and spleen were collected from adult White Leghorn hens. The stomach in developing stages was obtained from embryos, from incubation days 621, and from neonatal chickens from post-hatching days 012.
In Situ Hybridization
The sections were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.3) for 15 min and acetylated for 10 min with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0). The sections were prehybridized for 2 hr in a buffer containing 50% formamide, 0.1 M Tris-HCl (pH 8.0), 4 x SSC (1 x SSC = 150 mM NaCl and 15 mM sodium citrate), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.6 M NaCl, 0.25% sodium dodecyl sulfate (SDS), 200 µg/ml tRNA, 1 mM EDTA, and 10% dextran sulfate. Hybridization was performed at 42C for 10 hr in the prehybridization buffer supplemented with 10,000 cpm/µl of 35S-labeled oligonucleotide probes. The slides were washed at room temperature (RT) for 30 min in 2 x SSC containing 0.1% sarkosyl and twice at 55C for 40 min each in 0.1 x SSC containing 0.1% sarkosyl. The sections were dipped in Kodak NTB2 nuclear track emulsion (Kodak; Rochester, NY) and exposed for 1 month.
Cloning of Chicken Gut Chitinase
Total proventriculus RNA was isolated from a Leghorn chicken using TRIzol reagent (Gibco BRL; Grand Island, NY). First-strand cDNA synthesis was performed on 10 µg of total RNA using SuperScript II RNase H- reverse transcriptase (Gibco BRL) and a random primer. The first-strand cDNA was used as the template for polymerase chain reaction (PCR) amplification with Taq DNA polymerase (Promega) and a degenerated primer set. The primers were designed using human (AF290004) and bovine (AB051629) sequences. DNA sequence analysis was performed using the Big Dye terminator method (PE Applied Biosystems; Foster City, CA).
Northern Blotting and RT-PCR
Total RNA was separated in 1% agarose/formaldehyde gels, and transferred to nylon membranes (Amersham Pharmacia Biotech; Piscataway, NJ), which were hybridized with an [32P]-dCTP-labeled chicken gut chitinase probe (nucleotide residues 3321310) and exposed to X-ray film (Kodak).
To confirm the distribution of chicken gut chitinase cDNA, an RT-PCR experiment was performed using Taq DNA polymerase and first-strand cDNA as the template. The reaction was done using a specific primer set for chicken gut chitinase.
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Results |
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Expression of Gut Chitinase mRNA in Adult Mouse
Intense expression of gut chitinase mRNA was found only in the salivary gland and glandular stomach throughout the GI tract in adult mice (Fig 1 Fig 2 Fig 3 Fig 4). Other visceral organs, including the liver, pancreas, lung, kidney, adrenal gland, and urogenital organs, did not exhibit any significant expression. Of the three major salivary glands, only the parotid gland displayed intense expression, with selective localization in the glandular portion (acini), whereas the submandibular and sublingual glands completely lacked the signals (Fig 1). When tissue sections containing small salivary glands, such as the lingual gland, labial gland, and palatal gland, were hybridized, only the lingual gland of the serous type associated with circumvallate papillae, termed von Ebner's gland, expressed similarly intense transcription of gut chitinase mRNA (Fig 2). The specificity of hybridization was confirmed by consistent labeling using three non-overlapping antisense probes specific for mouse gut chitinase mRNA, and also by the disappearance of the signals when an excess dose of cold probes was added to the hybridization fluid (data not shown).
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The expression in the glandular stomach was analyzed in detail with special reference to pepsinogen C mRNA expression. Observation of serial sections alternatively hybridized for gut chitinase and pepsinogen C mRNAs revealed limited expression of chitinase mRNA in some pepsinogen C mRNA-expressing cells, thus identifiable as chief cells (Fig 3A and Fig 3B). Pepsinogen C mRNA-expressing cells occupied the basal half of gastric glands, from the bottom to the neck portion of the glands, but distribution of chitinase mRNA-expressing cells was restricted to the basal one-fourth of the gastric glands. Horizontally, pepsinogen cells were distributed throughout the oxyntic mucosa and extended to the proximal part of the pyloric antrum (Fig 4B). However, signals for chitinase mRNA in the oxyntic mucosa gradually decreased in intensity and extent approaching the pyloric mucosa; no significant signals were found in the pyloric antrum (Fig 4A).
Developmental Changes of Gut Chitinase mRNA Expression in Mouse Parotid Gland and Stomach
The first signals for gut chitinase mRNA in the developing parotid gland were detected at postnatal day (PD) 12, although they were weak and dispersed (Fig 5A). The intensity of chitinase mRNA expression sharply increased from PD 12 to PD 16 (Fig 5A5C). The first significant expression of chitinase mRNA in the developing stomach was found at PD 16. Only a small number of cells dispersed at the bottom of gastric glands were labeled (Fig 6A), in comparison to intense and broad expression of pepsinogen C mRNA (Fig 6B). Thereafter, the signals of chitinase mRNA gradually increased in intensity and extent and reached the adult level during PD 2024 (Fig 7). In contrast, intense signals for pepsinogen C mRNA already appeared at PD 0 (Fig 8). Throughout the developmental stages, no significant expression of gut chitinase mRNA was found in other parts of the GI tract or other visceral organs.
Chicken Gut Chitinase mRNA Expression in the Adult and During Ontogeny
Cloning and sequencing of chicken gut chitinase cDNA was carried out using RNA obtained from the adult chicken proventriculus (glandular stomach). We obtained the cDNA sequence of chicken gut chitinase from a combination of PCR products as described in Materials and Methods. The nucleotide sequence of the cloned cDNA contained an open reading frame 1449 bp long, and the open reading frame encoded a protein 482 amino acids long (CBPch04; accession no. AB071038). Overall sequence comparison of chicken gut chitinase with bovine, human, and mouse chitinases showed 70.2%, 75.8%, and 72.7% identity for the deduced amino acids, respectively (Fig 9). Chicken gut chitinase mRNA was detected in the proventriculus and liver by RT-PCR analysis (Fig 10). On the other hand, by Northern blotting analysis it was detected only in the proventriculus (Fig 11). These results indicated that chicken gut chitinase was expressed more abundantly in the proventriculus than in the liver.
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ISH analysis for chicken gut chitinase mRNA expression using two non-overlapping antisense oligonucleotide probes revealed its selective expression in the proventriculus and liver of the adult chicken (Fig 12A and Fig 12B). No signals were detectable in the lung, pancreas, kidney, spleen, and GI tract other than the proventriculus. The proventriculus had extremely intense signals. Oxyntico-peptic cells constituting the deep (proper) gastric glands expressed the mRNA, whereas duct portions of the deep gastric gland and superficial gastric gland lacked the signals (Fig 12A). Less intense but significant signals were found in the liver, where hepatocytes were labeled (Fig 12B).
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In the developing stages, the first significant signals of chicken chitinase mRNA in the proventriculus were found at embryonic day (ED) 7 (Fig 12C), and the signals gradually increased in intensity with development until the hatching day (Fig 12D and Fig 12E). Another sharp increase in intensity appeared around post-hatching day 2, showing the same intensity as seen in the adult chicken.
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Discussion |
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Mammalian gut chitinases have been identified in human, mouse, and cow (
Northern blotting analysis by
The present comparative staining for gut chitinase and pepsinogen C mRNAs showed that some chief cells expressed the chitinase mRNA. The distribution of pepsinogen C mRNA-expressing cells in the gastric glands of mice was in accordance with that reported in the rat (
Among the chitinase protein family, Ym1 and Ym2 are known to be expressed in the mammalian stomach (
The liver is another important source of gut chitinase in mammalian species. However, there is a clear species difference in the hepatic expression. Humans and rodents express chitinase mRNA in the salivary gland and stomach but not in the liver, whereas in the cattle only the liver expresses chitinase mRNA (
During the developing stages, it is reasonable that the first significant expression of gut chitinase appeared at PD 14 and 16 in the parotid gland and stomach, respectively, and rapidly increased in intensity in the following several days. This (possibly digestive) enzyme may be prepared just before the weaning period (about 21 days), whereas pepsinogen C is fully expressed at birth in the mouse (this study) and rat (
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Acknowledgments |
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Supported by a grant from the "Research for the Future" Program of the Japan Society for the Promotion of Science (JSPS-RFTF 97L00906).
Received for publication December 17, 2001; accepted February 20, 2002.
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