Inhibitory effect of a presenilin 1 mutation on the Wnt signalling pathway by enhancement of beta-catenin phosphorylation.

Mutations in the presenilin 1 (PS1) gene are the most common genetic factor underlying the development of early onset familial Alzheimer's disease (FAD). Accumulating evidence has shown that FAD-linked mutations of PS1 enhance the generation of amyloid-beta (1-42) protein. Recently, beta-catenin has been shown to interact with PS1. beta-catenin is essential for the Wnt signalling pathway. However, the biological significance of the interaction between beta-catenin and PS1 in this signalling pathway remains to be clarified. In this study, we investigated the effect of FAD-linked PS1 (M146L) mutation in the Wnt signalling pathway using the conditioned medium containing Wnt-3A. The expression of mutated PS1 inhibited the Wnt-3A-induced accumulation of beta-catenin. Chase analysis of beta-catenin in Wnt-3A-stimulated cells following cycloheximide treatment revealed that PS1 mutation enhanced the generation of the higher molecular mass form of beta-catenin, most likely, ubiquitinated beta-catenin. In addition, the expression of mutated PS1 elevated the level of phosphorylated beta-catenin, which is targeted to the ubiquitin/proteasome pathway. Thus, it appears that PS1 (M146L) mutation down-regulates the Wnt-3A-induced accumulation of beta-catenin due to an increase in the level of phosphorylated beta-catenin.

Signaling pathways leading to transcription and translation cooperatively regulate the transient increase in expression of c-Fos protein.

The mechanisms by which growth factors trigger signal transduction pathways leading to the regulation of c-Fos expression are of great interest. In this study we investigated the effect of hepatocyte growth factor (HGF/SF) and epidermal growth factor (EGF) on the expression of c-fos and its product, c-Fos, in human epithelial cell line MKN74. The expression level of c-Fos protein in HGF/SF-stimulated cells was 5--10-fold higher than that in EGF-stimulated cells, whereas the level of c-fos mRNA induced by HGF/SF was similar to that by EGF. The hyperphosphorylation of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), indicative of an increased number of translation initiation complexes, was detected only in HGF/SF-induced MKN74 cells. Activation of phosphatidylinositol-3'-OH kinase and FKBP12-rapamycin associated mammalian target of rapamycin (FRAP/mTOR) was observed after the treatment with HGF/SF. Pretreatment with an inhibitor of either one, i.e. LY294002 for phosphatidylinositol-3'-OH kinase or rapamycin for FRAP/mTOR, completely inhibited 4E-BP1 phosphorylation and decreased the c-Fos synthesis induced by HGF/SF down to the level found in EGF-induced cells. These results suggest that the phosphorylation of 4E-BP1 is stimulated by HGF/SF in a manner requiring both phosphatidy-linositol-3'-OH kinase-dependent and FRAP/mTOR-dependent pathways, thereby stimulating c-fos mRNA translation. Regulation of the translation process of c-fos mRNA in addition to the immediate activation of c-fos transcription is necessary for the transient increase in the level of c-Fos protein to stimulate cell proliferation.

The KMDB/MutationView: a mutation database for human disease genes.

The KMDB/MutationView is a graphical database of mutations in human disease-causing genes and its current version consists of nine category-based sub-databases including diseases of eye, heart, ear, brain, cancer, syndrome, autoimmunity, muscle and blood. The KMDB/MutationView stores mutation data of 97 genes involved in 87 different disease and is accessible through http://mutview.dmb.med. keio.ac.jp.

Characterization of a mutant E-cadherin protein encoded by a mutant gene frequently seen in diffuse-type human gastric carcinoma.

The cell-cell adhesion molecule E-cadherin plays an essential role in the maintenance and function of epithelial tissues. Altered expression of E-cadherin has been implicated in tumor invasion. Analysis of mutations of the human E-cadherin gene in gastric carcinoma of the diffuse type has revealed that deletion of exon 8 or 9 in its cDNA appears to be predominant. In this study, we carried out structural and functional analyses of a mutant form of E-cadherin in a cell line, HSC45-M2, established from a human signet ring-cell carcinoma. Although immunohistochemical analysis showed that the mutant cadherin was localized at cell-cell contact sites as usually seen with the wild type, these cells did not form compact colonies. HSC45-M2 cells expressed aberrant E-cadherin with an m.w. larger than that of the wild type. In these cells, we found deletion of the exon 9-intron 9 boundary including the splicing donor site in E-cadherin genomic DNA. RT-PCR indicated 2 transcripts, which appeared to be caused by the splicing defect. Northern blotting, however, showed that the transcript lacking exon 9 was predominantly detected in these cells. The electrophoretic mobilities on SDS-PAGE of the mutant E-cadherin protein in HSC45-M2 cells and the protein expressed from cDNA lacking exon 9 appeared identical. Analysis of the amino-terminal region of the mutant E-cadherin protein revealed that the cadherin was capable of becoming a mature form by removal of its amino-terminal peptide. However, the mutant E-cadherin was susceptible to trypsinization in the presence of Ca(2+), which is not the case for wild-type E-cadherin, suggesting that the mutant E-cadherin frequently found in diffuse-type gastric carcinoma may have lost its Ca(2+)-binding ability, leading to disruption of the tight cell-cell association.

Wnt-induced dephosphorylation of axin releases beta-catenin from the axin complex.

The stabilization of beta-catenin is a key regulatory step during cell fate changes and transformations to tumor cells. Several interacting proteins, including Axin, APC, and the protein kinase GSK-3beta are implicated in regulating beta-catenin phosphorylation and its subsequent degradation. Wnt signaling stabilizes beta-catenin, but it was not clear whether and how Wnt signaling regulates the beta-catenin complex. Here we show that Axin is dephosphorylated in response to Wnt signaling. The dephosphorylated Axin binds beta-catenin less efficiently than the phosphorylated form. Thus, Wnt signaling lowers Axin's affinity for beta-catenin, thereby disengaging beta-catenin from the degradation machinery.

Antagonistic regulation of cell migration by epidermal growth factor and glucocorticoid in human gastric carcinoma cells.

Epidermal growth factor (EGF) induced the disruption and scattering of colonies of TMK-1, a cell line derived from a human gastric carcinoma. A stimulatory action of EGF on cell migration was also observed as determined by a wound assay. However, these actions of EGF were inhibited if the cells were pretreated with dexamethasone, a synthetic glucocorticoid. Dexamethasone increased cell adhesion to collagen type IV and laminin, but not to poly-L-lysine and fibronectin. In contrast, EGF did not affect cell adhesion to these extracellular matrices whether dexamethasone was present or not. Dexamethasone enhanced the protein levels of both alpha1 and beta1 integrin subunits, and that of the alpha1 beta1 heterodimer. Further, flow cytometric analysis revealed that dexamethasone increased the expression of beta1 and alpha1 integrin subunits at the cell surface, whereas EGF increased expression of beta1 and alpha2 subunits at the cell surface. Antibodies against alpha1 and beta1 integrin subunits inhibited the increased cell adhesion seen in the presence of dexamethasone. An immunofluorescence study indicated that dexamethasone increased the formation of focal adhesions along the entire edges of cell colonies. In contrast, EGF led to the formation of focal adhesions preferentially at the cell front, and this EGF-induced preferential formation was not observed if the cells were pretreated with dexamethasone. These results suggest that glucocorticoid increased cell adhesion to the extracellular matrix via alpha1 beta1 integrin, and thereby antagonized EGF-induced cell migration.

Aberrant expression and phosphorylation of beta-catenin in human colorectal cancer.

The cytoplasmic domain of cadherins is known to associate with the intracellular proteins, catenins, which link cadherins to the actin-based cytoskeleton. In this study, we immunohistochemically investigated the expression of beta-catenin as well as E-cadherin and alpha-catenin in 86 human colorectal cancers, and we analysed their coexpression pattern and relationship to clinicopathological factors. In cancerous tissues, the frequency of reduced expression of beta-catenin (28 of 86, 33%) was similar to that of E-cadherin (19 of 86, 22%), but less than that of alpha-catenin (47 of 86, 55%). All three molecules were expressed strongly, as was the normal epithelium, in 36 cases (42%), whereas the rest (50 cases, 58%) showed reduction in one of the molecules. The reduction of beta-catenin expression was significantly correlated with dedifferentiation, Duke's stage, lymph node metastasis and liver metastasis. Next, we examined tyrosine phosphorylation in the protein complex immunoprecipitated with E-cadherin, as E-cadherin function is down-regulated by receptor-type tyrosine kinase in vitro. It was of interest that up-regulation of tyrosine phosphorylation of beta-catenin was more frequently observed in cancerous tissues than in the matching normal mucosa. These results suggest that beta-catenin may have important regulatory roles within an E-cadherin-mediated adhesion system in human colorectal cancers.

Cytoskeletal reorganization by soluble Wnt-3a protein signalling.

BACKGROUND: Wnt-3a is an intercellular signalling molecule that is involved in a variety of morphogenetic events. However, the molecular mechanisms underlying Wnt-3a signalling are poorly understood. We have sought to establish in vitro systems to assay the activity of this protein and investigate its biological roles. RESULTS: We prepared mouse L cells transfected with Wnt-3a cDNA, and found that their beta-catenin protein level was up-regulated. When conditioned medium (CM) was collected from cultures of the transfectants and added to nontransfected L cells, the beta-catenin level of the latter was also increased. Approximately 50% of the Wnt-3a proteins synthesized by the transfectants were secreted into the CM in a soluble form. These secreted Wnt-3a proteins formed an activity gradient in the environment surrounding the transfectants. Then, we studied whether Wnt-3a had any effect on cellular behaviour in vitro. When the CM containing Wnt-3a (W3a-CM) was added to cultures of C57MG mammary epithelial cells, their morphology was altered to exhibit closer intercellular contacts. Immunostaining for various adhesion and cytoskeletal proteins showed that the actin-microfilamental system was re-organized by the W3a-CM treatment. It induced a directional alignment of actin stress fibres and other actin-associated proteins. Moreover, villin, localized only at the perinuclear regions in untreated C57MG cells, was re-distributed to the leading edges of the cells, co-localizing with F-actin, in the presence of Wnt-3a. CONCLUSION: Our findings suggest that Wnt-3a protein, in the soluble form, can act to re-organize cytoskeletal structures.

Dissociation of c-fos induction and mitogen-activated-protein kinase activation from the hepatocyte-growth-factor-induced motility response in human gastric carcinoma cells.

The function of hepatocyte growth factor/scatter factor (HGF/SF) is to increase proliferation as well as to stimulate motility and disperse cell colonies of epithelial cells. In this study, we examined the motogenic and mitogenic responses of two human gastric carcinoma cell types, MKN7 and MKN74. Cell motility of both cell lines was markedly stimulated by HGF/SF. In contrast, HGF/SF stimulated cell growth of MKN74 cells, but did not stimulate growth of MKN7 cells. To address the cause of the difference in response of these cells, which may reflect some differences in signaling pathways downstream from the HGF/SF receptor, c-Met, we investigated the induction of the proto-oncogene c-fos. The level of c-fos mRNA increased and reached a maximum approximately 40 min after HGF/SF stimulation in MKN74 cells, and thereafter its level rapidly decreased. In contrast, the level of c-fos expression was very low irrespective of the stimulation in MKN7 cells. c-Fos protein was transiently induced only in MKN74 cells l h after treatment with HGF/SF, and its levels subsequently decreased. We subsequently examined the activation of mitogen-activated-protein kinase, which is a major mediator in the signaling pathway leading to the stimulation of c-fos transcription, after HGF/SF treatment in both cell lines. Mitogen-activated-protein kinase was markedly activated by this treatment in MKN74 cells, but was only slightly activated in MKN7 cells. These results suggest that although mitogen-activated-protein kinase activation and c-fos induction play an essential role in the signaling pathway leading to cell growth, they are not required for the motility response induced by HGF/SF.

Hepatocyte growth factor (HGF)-induced cell migration is negatively modulated by epidermal growth factor through tyrosine phosphorylation of the HGF receptor.

Hepatocyte growth factor (HGF) stimulated cell migration of human gastric carcinoma cell lines MKN1, MKN7, and MKN28. Epidermal growth factor (EGF) also stimulated the cell migration of these three cell lines. In MKN7 cells, HGF-stimulated cell migration was rather reduced in the presence of EGF, whereas such an observation was not made with MKN1 and MKN28 cells. Therefore, we compared the effect of EGF on HGF-stimulated HGF receptor phosphorylation in these cell lines. HGF induced a rapid tyrosine phosphorylation of the HGF receptor in all these cell lines. In MKN7 cells, the increased phosphorylation was further enhanced by EGF, although EGF alone did not affect tyrosine phosphorylation of the HGF receptor. In MKN1 and MKN28 cells, EGF did not influence tyrosine phosphorylation of the HGF receptor, whether HGF was present or not. The data presented here suggest that EGF negatively modulates the cellular response to HGF by increasing tyrosine phosphorylation of the HGF receptor in certain types of epithelial cells, e.g., MKN7 cells.

Beta-catenin expression in human cancers.

Cell-cell adhesion in tissue is mainly regulated by homotypic interaction of cadherin molecules, which are anchored to the cytoskeleton via cytoplasmic proteins, including alpha- and beta-catenin. Although we previously demonstrated that alpha-catenin is crucial for cadherin function in vivo, little is known about the role of beta-catenin. We examined the expression of beta-catenin in human carcinoma samples along with normal tissue (esophagus, stomach, and colon) by immunostaining using our antibody for beta-catenin. Normal epithelium strongly expressed beta-catenin. However, beta-catenin expression was frequently reduced in primary tumors of the esophagus (10 of 15, 67%), stomach (9 of 19, 47%), and colon (11 of 22, 50%). From an immunoprecipitation study, we found that beta-catenin forms a complex with E-cadherin not only in the normal epithelium but also in cancerous tissues. In coexpression patterns of E-cadherin and beta-catenin, 43 (77%) of the 56 tumors showed a similar expression of both molecules, whereas the other 13 tumors (23%) showed positive staining for E-cadherin and reduced expression of beta-catenin. These findings suggest that beta-catenin forms a complex with E-cadherin in vivo and down-regulation of beta-catenin expression is associated with malignant transformation.

Association of p120, a tyrosine kinase substrate, with E-cadherin/catenin complexes.

p120 was originally identified as a substrate of pp60src and several receptor tyrosine kinases, but its function is not known. Recent studies revealed that this protein shows homology to a group of proteins, beta-catenin/Armadillo and plakoglobin (gamma-catenin), which are associated with the cell adhesion molecules cadherins. In this study, we examined whether p120 is associated with E-cadherin using the human carcinoma cell line HT29, as well as other cell lines, which express both of these proteins. When proteins that copurified with E-cadherin were analyzed, not only alpha-catenin, beta-catenin, and plakoglobin but also p120 were detected. Conversely, immunoprecipitates of p120 contained E-cadherin and all the catenins, although a large subpopulation of p120 was not associated with E-cadherin. Analysis of these immunoprecipitates suggests that 20% or less of the extractable E-cadherin is associated with p120. When p120 immunoprecipitation was performed with cell lysates depleted of E-cadherin, beta-catenin was no longer coprecipitated, and the amount of plakoglobin copurified was greatly reduced. This finding suggests that there are various forms of p120 complexes, including p120/E-cadherin/beta-catenin and p120/E-cadherin/plakoglobin complexes; this association profile contrasts with the mutually exclusive association of beta-catenin and plakoglobin with cadherins. When the COOH-terminal catenin binding site was truncated from E-cadherin, not only beta-catenin but also p120 did not coprecipitate with this mutated E-cadherin. Immunocytological studies showed that p120 colocalized with E-cadherin at cell-cell contact sites, even after non-ionic detergent extraction. Treatment of cells with hepatocyte growth factor/scatter factor altered the level of tyrosine phosphorylation of p120 as well as of beta-catenin and plakoglobin. These results suggest that p120 associates with E-cadherin at its COOH-terminal region, but the mechanism for this association differs from that for the association of beta-catenin and plakoglobin with E-cadherin, and thus, that p120, whose function could be modulated by growth factors, may play a unique role in regulation of the cadherin-catenin adhesion system.

A truncated beta-catenin disrupts the interaction between E-cadherin and alpha-catenin: a cause of loss of intercellular adhesiveness in human cancer cell lines.

Cadherin cell adhesion molecules play an essential role in creating tight intercellular association and are considered to work as an invasion suppressor system of cancer cells. They form a molecular complex with catenins, a group of cytoplasmic proteins including alpha- and beta-catenins. While alpha-catenin has been demonstrated to be crucial for cadherin function, the role of beta-catenin is not yet fully understood. In this study, we analyzed the cadherin-catenin system in two human cell lines, HSC-39 and its putative subline HSC-40A, derived from a signet ring cell carcinoma of stomach. These cells grow as loose aggregates or single cells, suggesting that their cadherin system is not functional. In these cell lines, an identical 321-base pair in-frame mRNA deletion of beta-catenin was identified; this led to a 107-amino-acid deletion in the NH2-terminal region of the protein. Southern blot analysis disclosed a homozygous deletion in part of the beta-catenin gene. On the other hand, these cells expressed E-cadherin, alpha-catenin, and plakoglobin of normal size. Immunoprecipitation analyses showed that E-cadherin was coprecipitated with the mutated beta-catenin but not with alpha-catenin, and antibodies against beta-catenin did not copurify alpha-catenin. However, the recombinant fusion protein containing wild-type beta-catenin precipitated alpha-catenin from these cells. These results suggest that the dysfunction of E-cadherin in these cell lines is due primarily to its failure to interact with alpha-catenin, and that this defect results from the mutation in beta-catenin. Thus, it is most likely that the association between E-cadherin and alpha-catenin is mediated by beta-catenin, and that this process is blocked by NH2-terminal deletion in beta-catenin. These findings indicate that genetic abnormality of beta-catenin is one of the mechanisms responsible for loosening of cell-cell contact, and may be involved in enhancement of tumor invasion in human cancers.

Tyrosine phosphorylation of beta-catenin and plakoglobin enhanced by hepatocyte growth factor and epidermal growth factor in human carcinoma cells.

The effect of hepatocyte growth factor/scatter factor (HGF/SF) and epidermal growth factor (EGF) on cadherin-mediated adhesion of human carcinoma cells was studied. HGF/SF induced scattering of colonic adenocarcinoma HT29 and gastric adenocarcinomas MKN7 and MKN74 cells. Likewise, EGF induced scattering of HT29 and MKN7 cells. These cells expressed E-cadherin, which was concentrated at cell-cell contact sites. When the scattering of these cells was induced by HGF/SF or EGF, the E-cadherin concentration at cell-cell boundaries tended to decrease. Immunoblotting analyses, however, demonstrated that these growth factor treatments did not alter the expression of E-cadherin and E-cadherin-associated proteins, alpha- and beta-catenin and plakoglobin. beta-Catenin, plakoglobin and an unidentified 115-kDa molecule associated with E-cadherin were found to be phosphorylated at tyrosine residues, and these phosphorylations were enhanced by the growth factor treatments. These results suggest that HGF/SF and EGF may modulate the function of the cadherin-catenin system via tyrosine phosphorylation of cadherin-associated proteins.

Cadherin-dependent organization and disorganization of epithelial architecture.

Epithelial cell layers exhibit an ordered polarized architecture. However, such structures are disrupted during malignant transformation, which generally coincides with a loss of regulate cell growth. We are investigating how the cadherin cell adhesion system controls these processes. Cadherins form a molecular complex with alpha-catenin, and beta-catenin or plakoglobin at the cytoplasmic side in normal cells. Lung carcinoma PC9 cells express E-cadherin. Although they express other catenins, they lack alpha-catenin and cannot firmly aggregate, suggesting that their E-cadherin is inactive. Transfection of the PC9 cells with alpha-catenin cDNA leads to activation of the E-cadherin, inducing their compact aggregation. In these aggregates, an almost complete epithelial-specific architecture is organized, including the formation of microvilli and a junctional complex. We also studied the effect of hepatocyte growth factor/scatter factor (HGF/SF) on cell-cell contacts in keratinocyte cell lines, and found that this growth factor can disrupt desmosomal cell-cell contacts. HGF/SF, and also epidermal growth factor, enhance tyrosine phosphorylation of beta-catenin or plakoglobin in human carcinoma lines as they induce scattering of these cells. These findings suggest that the cadherin adhesion system is central in organizing epithelial structures and that tyrosine phosphorylation of catenins may modulate this organization process.

Stimulation of prostaglandin production by hepatocyte growth factor in human gastric carcinoma cells.

Hepatocyte growth factor (HGF), a protein with pleiotropic biological activity affecting cell growth and motility, was found to markedly activate prostaglandin production in human gastric carcinoma TMK-1 cells. HPLC analysis revealed that HGF stimulated the production of prostaglandin E2 (PGE2), which is the major prostaglandin produced in these cells. HGF maximally stimulated PGE2 production at a concentration of 10 ng/ml, and it was a more potent stimulator of PGE2 production than epidermal growth factor (EGF), which is known to stimulate prostaglandin production in various cell lines. The simultaneous addition of HGF and EGF caused no further stimulation of the PGE2 production observed in HGF-treated cells. We showed also that HGF increased the arachidonate release from TMK-1 cells, which release was completely suppressed by the addition of phospholipase A2 (PLA2) inhibitors. Further studies in vitro showed that HGF enhanced cellular activities of cytosolic PLA2 and cyclooxygenase 1.5-fold each. These results indicate that HGF stimulates prostaglandin production through increases in both cytosolic PLA2 and cyclooxygenase activities.

Dynamic control of cell-cell adhesion for multicellular organization.

Cadherin cell-cell adhesion molecules are associated with cytoskeletal proteins, including alpha and beta catenin, and plakoglobin. This cadherin-catenin complex plays an indispensable role in construction of ordered multicellular structures such as polarized epithelium. alpha-catenin is crucial for the cell binding function of cadherins; without it, cells cannot use the cadherin adhesion system for their adhesion. beta-catenin and plakoglobin possibly play more regulatory roles, as it was shown that their tyrosine phosphorylation correlated with modified cadherin activities. The expression of some cadherin-associated proteins is controlled by the wingless/Wnt-1 signal in embryos. These regulatory mechanisms of cadherin function and expression may be involved in dynamic control of cell-cell contacts during morphogenesis, and even in certain processes of cell growth and differentiation.

Proteolytic processing of the hepatocyte growth factor/scatter factor receptor by furin.

The hepatocyte growth factor/scatter factor (HGF/SF) receptor consists of an alpha- and a beta-subunit, which are derived from a single-chain precursor by endoproteolytic processing. The precursor is not proteolytically processed in LoVo colon carcinoma cells. The uncleaved receptor immunopurified from the cells was cleaved in vitro by furin. Furthermore, the HGF/SF receptor was proteolytically processed in LoVo cells transfected with furin cDNA. These results indicate that furin is a processing endoprotease for the HGF/SF receptor. Tyrosine autophosphorylation of the uncleaved receptor was induced by HGF/SF, and the growth of the cells expressing the uncleaved receptor was stimulated by HGF/SF, indicating that the proteolytic processing of the receptor is not essential for the signal transduction of HGF/SF.

Arachidonic acid-selective cytosolic phospholipase A2 is crucial in the cytotoxic action of tumor necrosis factor.

The role of phospholipase A2 (PLA2) in the cytotoxic action of tumor necrosis factor (TNF) was examined. Recently, we described the characterization of TNF-resistant sublines isolated from TNF-sensitive cell line L929 (Hayakawa, M., Oku, N., Takagi, T., Hori, T., Shibamoto, S., Yamanaka, Y., Takeuchi, K., Tsujimoto, M., and Ito, F. (1991) Cell Struct. Funct. 16, 333-340). The most remarkable features of these TNF-resistant sublines were the defects in TNF-stimulated arachidonate metabolism. In this study, we characterized the PLA2 activities of these sublines and found a significant decrease in the activity in one of the TNF-resistant sublines, C12 cells. The enzymological features, such as Ca2+ requirement and substrate specificity, suggested that the high molecular weight arachidonoyl-selective cytosolic PLA2 (cPLA2) was involved in the TNF action. Northern hybridization analysis demonstrated the reduced expression of high molecular weight cPLA2 in C12 cells. Furthermore, expression of a cloned cPLA2 cDNA in C12 cells increased the sensitivity of cells to the TNF cytotoxicity. These results indicate the crucial role of the high molecular weight cPLA2 in the TNF-induced cytolysis.