Interstitial cells of Cajal (ICC) are gastrointestinal (GI) pacemaker cells and intermediaries in enteric motor neurotransmission in the GI tract 12. 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 3. 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 4. Reduced numbers of ICC have also been reported in several GI motility disorders, such as chronic intestinal pseudo-obstruction 56, infantile hypertrophic pyloric stenosis 789, Hirschsprung's disease 101112, slow-transit constipation and certain forms of gastroparesis 1314.

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 1516. 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 1718.

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).

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 2122. p160 ROCK2, which is an isozyme of ROCK1 is a target for the small GTPase, Rho 23. 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 24. 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 25. This protein is supposed to belong to ADP-ribosyl cyclase family 26. Cellular RBP2 is an abundant 134-residue protein present in the small intestinal epithelium 27. 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 27.

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 28. RHAMM encodes a hyaluronan receptor protein 29. When hyaluronan binds to RHAMM, the phosphorylation of a number of proteins including the focal adhesion kinase pp125-FAK occurs 30. 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 31. It was reported that coproporphyria (CPO deficiency) patients showed constipation and abnormal colic that are main symptoms of this disease 32. 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 33.

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 1718.™@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 56, slow-transit constipation and certain forms of gastroparesis 1314, 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.

BP1/6C3: glutamyl aminopeptidase

BST1/BP3: bone marrow stromal antigen 1(alias ADP-ribosylcyclase 2 precursor)

CPO: coproporphyrinogen oxidase

DMP: deep muscular plexus

ESTs: expressed sequence tags

FISH: fluorescence in situ hybridization

GI: gastrointestinal

ICC: interstitial cells of Cajal

IC-DMP: ICC at the level of the deep muscular plexus

IC-IM: intramuscular ICC

IC-MY: ICC at the level of the myenteric plexus

MCM7: minichromosome maintenance 7

MY: myenteric plexus

p160 ROCK2: p160 Rho-associated, coiled-coil forming protein kinase 2

RBP2: retinol binding protein 2, cellular

RHAMM: hyaluronan-mediated motility receptor

RT-PCR: reverse transcription-polymerase chain reaction

SNP: single nucleotide polymorphism

None declared.

YD and IT carried out the molecular studies and the design of study and coordination. BAJP and CC participated in the molecular studies. SW, KS and MF participated in the design of study and coordination. All authors read and approved the final manuscript.