Differential gene expression in the murine gastric fundus lacking interstitial cells of Cajal
- Yataro Daigo†1, 2,
- Ichiro Takayama†1Email author,
- Bruce AJ Ponder2,
- Carlos Caldas2,
- Sean M Ward3,
- Kenton M Sanders3 and
- Masayuki A Fujino1
© Daigo et al; licensee BioMed Central Ltd. 2003
Received: 20 December 2002
Accepted: 10 June 2003
Published: 10 June 2003
The muscle layers of murine gastric fundus have no interstitial cells of Cajal at the level of the myenteric plexus and only possess intramuscular interstitial cells and this tissue does not generate electric slow waves. The absence of intramuscular interstitial cells in W/W V mutants provides a unique opportunity to study the molecular changes that are associated with the loss of these intercalating cells.
The gene expression profile of the gastric fundus of wild type and W/W V mice was assayed by murine microarray analysis displaying a total of 8734 elements. Queried genes from the microarray analysis were confirmed by semi-quantitative reverse transcription-polymerase chain reaction.
Twenty-one genes were differentially expressed in wild type and W/W V mice. Eleven transcripts had 2.0–2.5 fold higher mRNA expression in W/W V gastric fundus when compared to wild type tissues. Ten transcripts had 2.1–3.9 fold lower expression in W/W V mutants in comparison with wild type animals. None of these genes have ever been implicated in any bowel motility function.
These data provides evidence that several important genes have significantly changed in the murine fundus of W/W V mutants that lack intramuscular interstitial cells of Cajal and have reduced enteric motor neurotransmission.
Interstitial cells of Cajal (ICC) are gastrointestinal (GI) pacemaker cells and intermediaries in enteric motor neurotransmission in the GI tract [1, 2]. ICC express KIT/c-kit and depend on signalling via the gene product protein, KIT, a receptor tyrosine kinase which is essential for development and maintenance of the ICC phenotype . Mutations in the white-spotting locus (i.e. W/W V ) result in reduced KIT expression. These mutant animals develop few ICC at the level of the myenteric plexus (IC-MY) in the small intestine and the reduced ICC numbers is associated with a loss of slow wave activity. Intramuscular ICC (IC-IM) located in the stomach, lower esophageal and pyloric sphincters are absent in W/W V mutant animals . Reduced numbers of ICC have also been reported in several GI motility disorders, such as chronic intestinal pseudo-obstruction [5, 6], infantile hypertrophic pyloric stenosis [7–9], Hirschsprung's disease [10–12], slow-transit constipation and certain forms of gastroparesis [13, 14].
The association between motility disorders and loss of specific populations of ICC suggests that a more complete understanding of the molecular and cell biology of ICC networks within the gastrointestinal tract may help in understanding the etiology of some GI motor pathologies. The aim of the present study was to characterize genetic sequences that are expressed in ICC of the stomach that may encode important functional elements of the GI pacemaker/motor neurotransmission system. We pursued the hypothesis that such genes might show differential expression in the small intestines of wild type mice and W/W V mice [15, 16]. We previously identified fifteen known and novel genes that were differentially expressed in the small intestines of wild type and W/W V mice, which develop few IC-MY using a differential gene expression method [17, 18].
In the present study we hypothesized that there may also be differential expression of genes in the gastric fundus of W/W V mice, where IC-IM are lost. Our gene microarray analysis successfully identified 21 genes that were differentially expressed in the fundus of W/W V mice. The differential expression of these mice was confirmed by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).
Animals and Tissue Preparation
The use and treatment of experimental animals was approved by the Guideline governing Animal Experiment Committee at the University of Yamanashi School of Medicine and at the University of Nevada School of Medicine. Six adult male WBB6F1-+/+ mice (wild type) and age matched six adult male WBB6F1-W/W V mice, weighing 20 to 30 g, were purchased from Japan SLC Inc (Shizuoka, Japan). They were anesthetized with ether or carbon dioxide inhalation and sacrificed by cervical dislocation. For gene analyses, stomachs were removed from the animal and the mucosa with the attached submucosa rapidly sharp dissected from the tunica muscularis. Tissues were subsequently frozen in liquid nitrogen (-196 °C). Tissues were stored at -80 °C until isolation of RNA was preformed .
RNA Preparation and Microarray Data Analysis
Primer sets used for semi-quantitative RT-PCR.
CGA AAG CCT TGA GGT TGA AG
TAG GAA AAC AGG CGT CAC TG
GAA GAA AAG GCT GCA GAT CG
CAA GAG GCA AAG AGC AAT CC
CAC GAA TTG CAG GAC TAC CT
ACC TGC ACT GTA GGC TGA GT
GCC CAC TGG ATT GTG AAG AT
AGG AGA CTG GTC CAC ACC AC
AAG AAC CTG TCA AGC GTG GT
TCC AGG GTC ATC TGG AGT TC
AGA CTT GGT GGC AGA GGA GA
GCA GCT CAT GAC AGA ACA CC
CCT AAA GCA ACC CAA CCT GA
TAG CCT TAT GGG ACC TGG TG
GAT TTC TTG AGC TGG TGT CG
AAA ACC CTC TCG TGG GAT AG
CAG ATA GAG CAA GGG ATG GA
CTG AGC CCA AAC CAG TAG AA
GAC GAA GGA CCA AAA TGG AA
CGG TGA AAT CCA GGT CGT AG
AGC AAC AAC AGC TGG ACT TC
ACC TTC TGT TTG GTG CTG AG
TAT CGG CCT CAT CTA ACC AG
ATC TTC AAG CAG CAC CTG AC
CAT GCG ATA CTG GAA CAT GA
TGA CTC CAA ATA GCC CTC AG
TGG AGC AAG AGA GGA AAG TG
CTA GAC CTG AGC TTG CCT TG
ACG TGG AGA AAG TTC TCG TG
GCA ATA GTG TCA CCG AAT CC
GCC CTG GAG TTG AGA CTG TA
TGA CAA GCT GCA CAG TAA CC
AF031932 GCA GAA GGA GGA GCA GAG TG
GCA GTG ACG TCC CTC AGA CT
AAG TCT TTG TGT GGG CTG AG
AGG AAG CTT CGT CTC TCC AT
CAC CAG GAT GTC TGC CTA CT
CCG AGA TCA TGT TCT TCA CC
GAA GAC CAA GAT GGA GCT GA
CAG AAA GAT TCC CAT GAA CG
TTC ATC TGC TGC TCC TTC TC
TTA TAG ACC TTC CCG CAC AG
Microarray Analysis and Semi-quantitative RT-PCR
Analysis of gene expression in the fundus of wild type and W/W V mice
W/W V /wildtype ratio(a)
MCM7: DNA Replication Licensing Factor
p160 ROCK2: Rho-Associated Protein Kinase
BST1/BP3: ADP-Ribosyl Cyclase 2 Precursor
RBP2: retinol binding protein 2, cellular
BP1/6C3: Glutamyl Aminopeptitase
RHAMM: Hyaluronan-Mediated Motility Receptor
CPO: Coproporphyrinogen Oxidase
A comparison of differentially expressed genes from the fundus of W/W V mice using DNA microarray analysis revealed that the expression of eleven genes transcripts were significantly up-regulated in W/W V mice, whereas ten genes transcripts were dramatically suppressed in these animals. We confirmed these results with semi-quantitative RT-PCR of tissues from six each of wild type and W/W V mice. Data obtained from these experiments suggest that expression of the genes were specifically regulated in W/W V mice.
The eleven genes that were up-regulated in the gastric fundus of W/W V mice were identified as MCM7, p160 ROCK2, BST1/BP3, RBP2, and another seven unannotated transcripts. MCM7 is a mammalian homologue of the yeast nuclear protein MCM2/CDC47, which is thought to play an important role in two crucial steps of the cell cycle, namely, onset of DNA replication and cell division [21, 22]. p160 ROCK2, which is an isozyme of ROCK1 is a target for the small GTPase, Rho . ROCK2 is a serine/threonine kinase that regulates cytokinesis, smooth muscle contraction, the formation of actin stress fibers and focal adhesions, and the activation of the FOS serum response element . The up-regulation of p160 ROCK2 may have a compensating effect for the loss of ICC-dependent mechanisms in the gastric fundus. The BST1/BP3, a bone marrow stromal cell surface antigen, is a variably glycosylated glycosyl-phosphatidylinositol (GPI)-linked molecule that is selectively expressed by early B and T lineage cells and a discrete subpopulation of reticular cells in the peripheral lymphoid organs. It is also expressed on the brush border of intestinal epithelial cells, the luminal surface of renal collecting tubules and mature myeloid cells . This protein is supposed to belong to ADP-ribosyl cyclase family . Cellular RBP2 is an abundant 134-residue protein present in the small intestinal epithelium . It is thought to participate in the uptake and/or intracellular metabolism of vitamin A and belong to a protein family that contains liver fatty acid-binding protein. Vitamin A is a fat-soluble vitamin necessary for growth, reproduction, differentiation of epithelial tissues, and vision. Mammals depend on intestinal absorption of this vitamin for their survival. RBP2, which is confined largely to the small intestinal enterocyte, probably plays an important role in the intestinal absorption and/or metabolism of vitamin A .
We also confirmed the down-regulation of ten genes in W/W V mice: BP1/6C3, RHAMM, CPO, and another seven unannotated ESTs. The murine β-lymphocyte differentiation antigen, BP1/6C3 was characterized as glutamyl aminopeptidase, which is reported to serve as cell-differentiation marker of lymphomyelocytic lineages and may be involved in cell activation, signal transduction, and cell-matrix adhesion. It is also expressed by capillary endothelial cells, placenta, and epithelial cells of the intestine and proximal renal tubules . RHAMM encodes a hyaluronan receptor protein . When hyaluronan binds to RHAMM, the phosphorylation of a number of proteins including the focal adhesion kinase pp125-FAK occurs . This is a necessary step for disassembly of focal contacts and subsequent motility. CPO is the sixth enzyme of the heme biosynthetic pathway. This soluble protein is localized in the intermembrane space of mitochondria and catalyzes the conversion of two propionate groups at positions two and four of coproporphyrinogen III to two vinyl groups of protoporphyrinogen IX . It was reported that coproporphyria (CPO deficiency) patients showed constipation and abnormal colic that are main symptoms of this disease . Marked elevation of coproporphyria in the feces differentiated the condition from the Swedish type in which stool porphyrins are usually normal and from variegate porphyria in which both coproporphyrin and protoporphyrinogen fractions are increased in the stool .
In the 21 genes identified, we determined the subcellular localization and human chromosomal mapping of the 7 known genes using the SOURCE program http://source.stanford.edu (Table 2). These genes showed a variety of cellular localization including 3 membrane, 2 cytoplasmic, 1 mitochondrial and 1 nuclear proteins. Further analyses of these genes might enable us to elucidate not only their relationship with the KIT, a receptor tyrosine kinase, but also the molecular aspects of GI pacemaker system.
We previously identified fifteen genes that were differentially expressed in the small intestines of wild type and W/W V mice which develop few IC-MY using a differential gene expression method [17, 18].™@None of these 15 genes were found in the list of 21 genes that were differentially expressed in the gastric fundus of wild type and W/W V mice which develop few IC-IM using a cDNA microarray. As we confirmed differential gene expression of KIT between the small intestines/gastric fundic tissues of wild type and those of W/W V mutant mice by semi-quantitative RT-PCR, our experimental system could detect genetic aberration in W/W V mice. Therefore our results might reflect the difference of the cellular entity of two types of ICCs, IC-MY and IC-IM, or the difference of the cellular composition between small intestine and fundus. To provide conclusive evidence, which support these speculations, expression profile using purified mRNA from isolated single interstitial cells might be very useful in the next study.
At the present time we do not know whether these genes are important to the function of the gastric pacemaker/neurotransmission apparatus or were down- or up-regulated as a result of the loss of ICC. These data, however, provide clear systemic genetic evidence that several important proteins that have roles in cell cycle, cytokinesis and formation of cytoskeleton, cellular metabolism, oxygen metabolism, cell adhesion, and development and differentiation of gut cells are significantly changed in the gastric fundus of W/W V mice. Generation of transgenic animals that show tissue-specific overexpression of the candidate genes, as well as the gene knockout animals might be helpful for elucidating the role of each gene in gastrointestinal motility.
The discovery of an entire human and mouse genes through the genome project is supposed to revolutionize biological medicine including molecular diagnosis of various diseases and development of novel treatment. The information combined with high throughput technology such as DNA microarray and SNP typing analysis will accelerate discovery of genes susceptible to or causing various diseases and contribute to screening of novel drugs that target these disease-gene products. In this sense, the effort of the molecular profiling project in which we attempt to discover genetic aberration in animal disease models such as W/W V mice will generate very variable resources for further elucidation of motility disorders. As reduced numbers of ICC have also been reported in several GI motility disorders, such as chronic intestinal pseudo-obstruction [5, 6], slow-transit constipation and certain forms of gastroparesis [13, 14], these gene information should aid the development of novel molecular-targeted therapies for these disorders, and may also identify diagnostic molecular markers for these disorders.
We have identified twenty-one genes that encode functional proteins that are significantly up- or down-regulated in the tunica muscularis of the gastric fundus of W/W V mice. Considering that none of these genes has been implicated in any aspect of GI motility, we suggest that many unknown genes could be involved in the cellular changes that lead to motility disorders associated with the loss of ICC. Gene microarray analysis is an effective method for screening the changes that occur in the expression patterns of genes in response to spontaneous or genetic mutations that lead to the loss of a specific cell type. Application of this technique may allow recognition of patterns of gene expression that are common to the several motility disorders in which ICC are lost, thus providing new insights into the molecular mechanisms responsible for the selective loss of ICC populations.
List of abbreviations
bone marrow stromal antigen 1(alias ADP-ribosylcyclase 2 precursor)
deep muscular plexus
expressed sequence tags
fluorescence in situ hybridization
interstitial cells of Cajal
ICC at the level of the deep muscular plexus
ICC at the level of the myenteric plexus
minichromosome maintenance 7
- p160 ROCK2:
p160 Rho-associated, coiled-coil forming protein kinase 2
retinol binding protein 2, cellular
hyaluronan-mediated motility receptor
reverse transcription-polymerase chain reaction
single nucleotide polymorphism
Supported by 08457165 (to MF) from the Japanese Ministry of Education, Science, Sports and Culture, JAPAN and DK 57236 (to SW) and PO1 DK41315 (to SW and KS) from the National Institutes of Health, USA.
- Ward SM, Burns AJ, Torihashi S, Sanders KM: Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol (Lond). 1994, 480: 91-97.View ArticleGoogle Scholar
- Burns AJ, Herbert TM, Ward SM, Sanders KM: Interstitial cells of Cajal inhibit neurotransmission in the stomach. Proc Natl Acad Sci USA. 1996, 93: 12008-12013. 10.1073/pnas.93.21.12008.View ArticlePubMedPubMed CentralGoogle Scholar
- Huizinga JD, Thuneberg L, Kluppel M, Malysz J, Mikkelesen HB, Bernstein A: W/kit gene required for interstitial cells of Cajal and for intestinal pacemaker activity. Nature (Lond). 1995, 373: 347-349. 10.1038/373347a0.View ArticleGoogle Scholar
- Ward SM, Beckett EAH, Wang XY, Baker F, Khoyi M, Sanders KM: Interstitial cells of Cajal mediate enteric excitatory neurotransmission in the murine fundus. J Neurosci. 2000, 20: 1393-1403.PubMedGoogle Scholar
- Takayama I, Kojima Y, Ohtsuka H, Sato T, Fujino MA: Pathology of human idiopathic intestinal pseudo-obstruction and W/W v mice in gut motility: A gut pacemaker disease?. Gut. 1995, 37 (Suppl): A85-Google Scholar
- Isozaki K, Hirota S, Miyagawa J, Taniguchi M, Shiomura Y, Matsuzawa Y: Deficiency of c-kit+ cells in patients with a myopathic form of chronic idiopathic intestinal pseudo-obstruction. Am J Gastroenterol. 1997, 92: 332-334.PubMedGoogle Scholar
- Vanderwinden JM, Liu H, Menu R, Conreur JL, De Laet MH, Vanderhagen JJ: The pathology of infantile hypertrophic pyloric stenosis after healing. J Pediatr Surg. 1996, 31: 1530-1534.View ArticlePubMedGoogle Scholar
- Vanderwinden JM, Liu H, DeLaet MH, Vanderhagen JJ: Study of the interstitial cells of Cajal in infantile hypertrophic pyloric stenosis. Gastroenterol. 1996, 111: 279-288.View ArticleGoogle Scholar
- Yamataka A, Fujiwara T, Kato Y, Okazaki T, Sunagawa M, Miyano T: Lack of intestinal pacemaker (C-KIT-positive) cells in infantile hypertrophic pyloric stenosis. J Pediatr Surg. 1996, 31: 96-98.View ArticlePubMedGoogle Scholar
- Yamataka A, Kato Y, Tibboel D: A lack of intestinal pacemaker (c-kit) in aganglionic bowel of patients with Hirschsprung's disease. J Pediatr Surg. 1995, 30: 441-444.View ArticlePubMedGoogle Scholar
- Yamataka A, Ohshiro K, Kobayashi H, Fujiwara T, Sunagawa M, Miyano T: Interstitial C-KIT+ cells and synapse in allied Hirschsprung's disorders. J Pediatr Surg. 1997, 30: 1069-1074.View ArticleGoogle Scholar
- Vanderwinden JM, Liu H, DeLaet NH, Vanderhagen JJ: Interstitial cells of Cajal in human colon and in Hirschsprung's disease. Gastroenterol. 1996, 111: 901-910.View ArticleGoogle Scholar
- He CL, Burgart L, Wang L, Pemberton J, Young-Fadok L, Szurszewski J, Farrugia G: Decreased interstitial cell of Cajal volume in patients with slow-transit constipation. Gastroenterol. 2000, 118: 14-21.View ArticleGoogle Scholar
- Ordog T, Takayama I, Cheung WKT, Ward SM, Sanders KM: Remodeling of networks of interstitial cells of Cajal in a murine model of diabetic gastroparesis. Diabetes. 2001, 49: 1731-1739.View ArticleGoogle Scholar
- Takayama I, Daigo Y, Kojima Y, Fujino MA: Gastrointestinal pacemaker system. Nippon Shoukakibyo Gakkai Zasshi. 2001, 98: 922-934.Google Scholar
- Takayama I, Horiguchi K, Daigo Y, Mine T, Fujino MA, Ohno S: The interstitial cells of Cajal and a gastrointestinal pacemaker system. Arch Histol Cytol. 2002, 65: 1-26.View ArticlePubMedGoogle Scholar
- Takayama I, Daigo Y, Ward SM, Sanders KM, Yamanaka T, Fujino MA: Differential gene expression in the small intestines of wildtype and W/W V mice. Neurogastroenterol Motil. 2001, 13: 163-168. 10.1046/j.1365-2982.2001.00256.x.View ArticlePubMedGoogle Scholar
- Takayama I, Daigo Y, Ward SM, Sanders KM, Walker RL, Horowitz B, Yamanaka T, Fujino MA: Novel human and mouse genes encoding an acid phosphatase family member and its down regulation in W/W V mouse jejunum. Gut. 2002, 50: 790-796. 10.1136/gut.50.6.790.View ArticlePubMedPubMed CentralGoogle Scholar
- Liang P, Pardee AB: Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science. 1992, 257: 967-971.View ArticlePubMedGoogle Scholar
- Ljubimova JY, Khazenzon NM, Chen Z, Neyman YI, Turner L, Riedinger MS, Black KL: Gene expression abnormalities in human glial tumors identified by gene array. Int J Oncol. 2001, 18: 287-295.PubMedGoogle Scholar
- Nakatsuru S, Sudo K, Nakamura Y: Isolation and mapping of a human gene (MCM2) encoding a product homologous to yeast proteins involved in DNA replication. Cell Genet. 1995, 68: 226-230.View ArticleGoogle Scholar
- Labib K, Tercero JA, Diffley JFX: DNA replication fork progression requires uninterrupted MCM2-7 function. Science. 2000, 288: 1643-1647. 10.1126/science.288.5471.1643.View ArticlePubMedGoogle Scholar
- Takahashi N, Tuiki H, Saya H, Kaibuchi K: Localization of the gene coding for ROCK II/Rho kinase on human chromosome 2p24. Genomics. 1999, 55: 235-237. 10.1006/geno.1998.5344.View ArticlePubMedGoogle Scholar
- Maekawa M, Ishizaki T, Boku S, Watanabe N, Fujita A, Iwamatsu A, Obinata T, Ohashi K, Mizuno K, Narumiya S: Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science. 1999, 285: 895-898. 10.1126/science.285.5429.895.View ArticlePubMedGoogle Scholar
- Dong C, Wang J, Neame P, Cooper MD: The murine BP-3 gene encodes a relative of the CD38/NAD glycohydrolase family. Int Immunol. 1994, 6: 1353-1360.View ArticlePubMedGoogle Scholar
- Itoh M, Ishihara K, Tomizawa H, Tanaka H, Kobune Y, Ishikawa J, Kaisho T, Hirano T: Molecular cloning of murine BST-1 having homology with CD38 and Aplysia ADP-ribosyl cyclase. Biochem Biophys Res Commun. 1994, 203: 1309-1317. 10.1006/bbrc.1994.2325.View ArticlePubMedGoogle Scholar
- Demmer LA, Birkenmeier EH, Sweetser DA, Levin MS, Zollman S, Sparkes RS, Mohandas T, Lusis AJ, Gordon JI: The cellular retinol binding protein II gene: sequence analysis of the rat gene, chromosomal localization in mice and humans, and documentation of its close linkage to the cellular retinol binding protein gene. J Biol Chem. 1987, 262: 2458-2467.PubMedGoogle Scholar
- Wu Q, Li L, Cooper MD, Pierres M, Gorvel JP: Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3. Proc Nat Acad Sci. 1991, 88: 676-680.View ArticlePubMedPubMed CentralGoogle Scholar
- Hardwick C, Hoare K, Owens R, Hohn HP, Moore D, Cripps V, Austen L, Nance DM, Turley EA: Molecular cloning of a novel hyaluronan receptor that mediates tumor cell motility. J Cell Biol. 1992, 117: 1343-1350.View ArticlePubMedGoogle Scholar
- Hall CL, Wang C, Lange LA, Turley EA: Hyaluronan and the hyaluronan receptor RHAMM promote focal adhesion turnover and transient tyrosine kinase activity. J Cell Biol. 1994, 126: 575-588.View ArticlePubMedGoogle Scholar
- Martasek P, Camadro JM, Delfau-Larue MH, Dumas JB, Montagne JJ, De Verneuil H, Labbe P, Grandchamp B: Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase. Proc Nat Acad Sci. 1994, 91: 3024-3028.View ArticlePubMedPubMed CentralGoogle Scholar
- Macy JA, Gilroy J, Perrin JC: Hereditary coproporphyria: a imitator of multiple sclerosis. Arch Phys Med Rehabil. 1991, 72: 703-704.PubMedGoogle Scholar
- Barnes HD, Whittaker N: Hereditary coproporphyria with acute intermittent manifestations. Brit Med J. 1965, 2: 1102-1104.View ArticlePubMedPubMed CentralGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-230X/3/14/prepub
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