Kenji Watabe

The Spermatogenic Ig Superfamily/Synaptic Cell Adhesion Molecule Mast-Cell Adhesion Molecule Promotes Interaction with Nerves

Nerve-mast cell interaction is involved in both homeostatic and pathologic regulations. The molecules that sustain this association have not been identified. Because synaptic cell adhesion molecule (SynCAM), alternatively named spermatogenic Ig superfamily (SgIGSF), is expressed on both nerves and mast cells and because it binds homophilically, this molecule may be a candidate. To examine this possibility, mast cells with or without SgIGSF/SynCAM were cocultured with superior cervical ganglion neurons that express SgIGSF/SynCAM, and the number of mast cells attached to neurites was counted. The attachment of mast cells with SgIGSF/SynCAM, i.e., bone marrow-derived mast cells (BMMC) from wild-type mice, was inhibited dose-dependently by blocking Ab to SgIGSF/SynCAM. Mast cells without SgIGSF/SynCAM, i.e., BMMC from microphthalmia transcription factor-deficient mice and BMMC-derived cell line IC-2 cells, were defective in attachment to neurite, and transfection with SgIGSF/SynCAM normalized this. When the nerves were specifically activated by scorpion venom, one-quarter of the attached IC-2 cells mobilized Ca2+ after a few dozen seconds, and ectopic SgIGSF/SynCAM doubled this proportion. At points of contact between neurites and wild-type BMMC, SgIGSF/SynCAM was locally concentrated in both neurites and BMMC. SgIGSF/SynCAM on mast cells appeared to predominantly mediate attachment and promote communication with nerves.

Disturbed gastrointestinal motility and decreased interstitial cells of Cajal in diabetic db/db mice

Background and Aim:  Diabetes mellitus (DM) often causes gastrointestinal dysmotility. Interstitial cells of Cajal (ICC), which express c‐kit receptor tyrosine kinase (KIT), are considered the pacemaker cells for gastrointestinal movement. The present study was designed to determine the role of ICC in the pathogenesis of gastroenteropathy in type 2 DM.

Distinct role for c-kit receptor tyrosine kinase and SgIGSF adhesion molecule in attachment of mast cells to fibroblasts.

Binding of stem cell factor (SCF) to c-kit receptor tyrosine kinase (KIT) transduces signals essential for mast cell development via several pathways including activation of phosphatidylinositol 3-kinase (PI3-K). When cultured mast cells (CMCs) are cocultured with fibroblasts expressing membrane-bound SCF, CMCs with normal KIT adhere to fibroblasts and proliferate, whereas CMCs lacking cell surface expression of KIT do neither. Spermatogenic immunoglobulin superfamily (SgIGSF) was identified as another molecule that participates in mast cell adhesion to fibroblasts. Since the IC-2 mast cell line expressed neither KIT nor SgIGSF, the effect of ectopic expression of KIT or SgIGSF on the adhesion of IC-2 cells was examined. Three forms of KIT with the normal ectodomain were used: wild-type (KIT-WT) and two mutant types with a phenylalanine substitution at the tyrosine residue 719 (KIT-Y719F) or 821 (KIT-Y821F). KIT-Y719F does not activate PI3-K, whereas KIT-Y821F does. Firstly, KIT or SgIGSF was expressed singly in IC-2 cells. All three forms of KIT increased the adhesion level of IC-2 cells, whereas SgIGSF did not. Secondly, SgIGSF was coexpressed with one of the three forms of KIT. Coexpression of SgIGSF with KIT-WT or KIT-Y821F increased the adhesion level more markedly than was achieved by KIT-WT or KIT-Y821F alone. The effect was abolished by an antibody that blocks SCF–KIT interaction. In contrast, coexpression of SgIGSF with KIT-Y719F did not increase the adhesion level induced by KIT-Y719F alone. In adhesion of mast cells to fibroblasts, KIT appeared to behave as an adhesion molecule and as an activator of other adhesion molecules through phosphorylating PI3-K.

Cloning of a soluble isoform of the SgIGSF adhesion molecule that binds the extracellular domain of the membrane-bound isoform

SgIGSF (spermatogenic immunoglobulin superfamily) is a recently identified intercellular adhesion molecule of the immunoglobulin superfamily. In a mast-cell cDNA library, we found a clone that resulted from the retention of intron 7 within the mature SgIGSF message. This clone was predicted to encode a soluble isoform of SgIGSF (sSgIGSF) with 336 amino-acid residues because its open reading frame ended just before the transmembrane domain. We constructed a plasmid expressing sSgIGSF fused to the human IgG Fc fragment at its C-terminus (sSgIGSF-Fc), and transfected it into COS-7 cells. The fusion protein was readily detectable in the culture supernatant. Solid-phase binding assay showed that sSgIGSF interacted directly the extracellular domain of membrane-bound SgIGSF (mSgIGSF). We next examined whether this interaction inhibited homophilic binding of mSgIGSF by aggregation assays using L cells that did not express mSgIGSF. A stable L-cell clone that overexpressed mSgIGSF aggregated with each other but not with mock-transfected L cells, indicating that a homophilic interaction of mSgIGSF mediated the aggregation. Addition of sSgIGSF-Fc inhibited the aggregation of L cells overexpressing mSgIGSF in a dose-dependent manner. Moreover, FACScan analyses revealed the specific binding of sSgIGSF-Fc to mSgIGSF expressed in L cells. Binding of sSgIGSF-Fc to mSgIGSF appeared to inhibit homophilic interactions of mSgIGSF.

Structure, expression and chromosome mapping of MLZE, a novel gene which is preferentially expressed in metastatic melanoma cells

We isolated a novel gene, termed MLZE, from a B16‐BL6 cDNA library after subtraction of B16‐F10 mRNA. Expression levels of mouse MLZE (mMLZE) increased in accordance with metastatic ability of B16 melanoma sublines. Human homolog of mMlze (hMlze) contained one leucine zipper structure and two potential nuclear localizing signals. Northern blot analysis of multiple human tissues showed that hMLZE was expressed primarily in trachea and spleen. We mapped the hMLZE gene (by fluorescence in situ hybridization) to 8q24.1‐2, which contains the c‐myc gene and is often amplified in malignant melanoma. Immunohistochemistry revealed that the number of hMlze‐positive cases was significantly larger in Clark levels III, IV and V melanomas (6/11=55%) than in Clark levels I and II melanomas (2/15=13%). In two cases of hMlze‐positive melanomas, the strength of hMlze staining increased substantially in the deep component of the tumor. Considering that melanomas above Clark level II are more metastatic than those below Clark level III, these findings suggested that MLZE is one of the genes whose expression is upregulated during the course of acquisition of metastatic potential in melanoma cells.

E-cadherin Gene Promoter Hypermethylation in H. pylori-Induced Enlarged Fold Gastritis

Background:  Promoter hypermethylation of E‐cadherin plays an important role on gastric carcinogenesis. We have previously reported that the odds ratio for gastric carcinoma and the prevalence of diffuse‐type early gastric carcinoma in Helicobacter pylori‐induced enlarged fold gastritis increased with increasing fold width. Thus, we examined E‐cadherin methylation in gastric mucosa from H. pylori‐induced enlarged fold gastritis before and after H. pylori eradication. Moreover, we analyzed the mechanism of H. pylori infection‐induced E‐cadherin hypermethylation.

Increased Expression of a Nucleolar Nop5/Sik Family Member in Metastatic Melanoma Cells : Evidence for Its Role in Nucleolar Sizing and Function

F10 and BL6 cells of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells can metastasize to lungs after subcutaneous injection. Differences in gene expression between the two cell lines were examined, and a greater expression of the Sik-similar protein (Sik-SP) gene was found in BL6 cells. Structurally, Sik-SP belongs to the nucleolar Nop5/Sik family whose members play central roles in ribosome biogenesis; however, the function of Sik-SP has not been examined. Cytology with green fluorescent protein-fused proteins showed that Sik-SP was localized to the nucleolus. To examine whether Sik-SP is involved in ribosome biogenesis, two parameters were measured: magnitude of ribosomal RNA synthesis per nucleus and magnitude of protein production from the same amount of mRNA of an exogenous luciferase gene. Both values and, in addition, nucleolar size were larger in COS-7 monkey kidney cells overexpressing Sik-SP and BL6 cells than in mock-transfected COS-7 and F10 cells, respectively. Sik-SP seemed to promote ribosome biogenesis in the nucleolus. Furthermore, the expression of Sik-SP seemed to confer a greater cell growth response to serum, because such a response was greater in BL6 cells and F10 cells overexpressing Sik-SP than in untreated and mock-transfected F10 cells. Sik-SP may render melanoma cells more competent to survive through augmenting the activity of nucleolus.

A Truncated Isoform of the Protein Phosphatase 2A B56γ Regulatory Subunit May Promote Genetic Instability and Cause Tumor Progression

F10, a subline of the B16 mouse melanoma cell line, is itself the parent of the more metastatic BL6 line. BL6 cells differ from F10 cells by an alteration of the gene encoding the B56gamma regulatory subunit of protein phosphatase 2A (PP2A), which results in the expression of a truncated variant of the subunit (Deltagamma1). PP2A is involved in regulating the cell-cycle checkpoint and we found that the checkpoint in BL6 cells is aberrant when the Deltagamma1 protein is expressed. That is, although Deltagamma1 protein levels in cultured BL6 cells are low and these cells do not show an altered checkpoint on gamma-irradiation, irradiated footpad BL6 tumor cells show both a marked increase in Deltagamma1 levels and more extensive polyploidy and less apoptosis than F10 cells. These observations were reproduced with Deltagamma1 gene-transfected F10 cells (F10(Deltagamma1)). Deltagamma1 expression and an aberrant checkpoint are also associated with a higher metastatic ability because irradiated F10(Deltagamma1) tumors metastasized much more frequently than F10 tumors, which rarely metastasized whether irradiated or not. Nonirradiated F10(Deltagamma1) tumors, which do not express Deltagamma1 protein, had similarly low rates of metastasis. The greater metastatic ability of irradiated F10(Deltagamma1) tumors also correlated with the acquisition of many more genomic alterations. Thus, it seems that Deltagamma1 expression may damage the checkpoint, which may then allow the acquisition of genetic alterations that promote metastasis. These observations support the notion that mechanisms promoting the genetic instability of tumors could also aid tumor progression from the nonmetastatic to the metastatic state.

Annexin VII as a novel marker for invasive phenotype of malignant melanoma.

Both F10 and BL6 sublines of B16 mouse melanoma cells are metastatic after intravenous injection, but only BL6 cells are metastatic after subcutaneous injection. While examining the genetic difference between the two sublines, we found a marked reduction of annexin VII expression in BL6 cells. In addition, fusion cell clones of both sublines, were as poorly metastatic as F10 cells after subcutaneous injection, and contained the annexin VII message as abundantly as F10 cells. Hence, we examined whether the annexin VII expression was correlated with the less malignant phenotype of clinical cases by immunohistochemistry. Immunoreactivities to anti‐annexin VII antibody in melanoma cells were evaluated quantitatively by using skin mast cells as an internal positive control. Eighteen patients with malignant melanoma were divided into two groups: lymph node metastasis‐negative and positive groups. The ratio of numbers of patients positive versus negative to the antibody was significantly larger in the former than in the latter group. These results not only indicated that annexin VII serves as a marker for less invasive phenotype of malignant melanoma, but also suggested a possible role of annexin VII in tumor suppression.

Is Obesity a New Risk Factor for Gastritis

Obesity has become a major concern among gastroenterologists due to its large influence on gastrointestinal and hepatic diseases: reflux esophagitis, pancreatitis, gallstone disease, liver fibrosis, and neoplastic tumors of the esophagus, pancreas, and colon. Studies of morbid obese subjects undergoing bariatric surgery have revealed that obesity is related with an increased prevalence of endoscopic and histologic gastritis. A recent study of health check-up subjects demonstrated an association of obesity with endoscopic gastritis and gastric ulcers. We recently investigated the underlying mechanisms of the effects of obesity on endoscopic gastritis in subjects undergoing health check-up examination, and demonstrated that adiponectin, a bioactive molecule released from visceral fat, could be a protective factor of endoscopic gastritis. We would like to propose a new category of gastritis, obesity-related gastritis, which could become dominant in the near future.