PubMed Health⌕ Search

Biomedical subjects

Tsuyoshi Saito

Publications and source records attributed to Tsuyoshi Saito.

At least 19 recordsLinked to original sources
PubMed2006

Inhibitory effects of an orexin-2 receptor antagonist on orexin A- and stress-induced ACTH responses in conscious rats.

Orexins, recognized for their diverse functions in sleep/wakefulness/arousal and appetite regulation, may play provocative roles in stress response. Although the PVN of the hypothalamus expresses an abundance of orexin-2 receptor (OX-2R), the involvement of OX-2R in regulating ACTH response to stress remains unclear. To address this, we examined effects of a selective antagonist to OX-2R (N-{(1S)-1-[6,7-dimethoxy-3,4-dihydro-2(1H)-isoquinolinyl]carbonyl}-2,2-dimethylpropyl)-N-{4-pyridinylmethyl}amine upon plasma ACTH concentrations after administration of orexin A and swimming stress. Increases in ACTH levels with orexin A or swimming stress were attenuated with prior administration of an OX-2R antagonist. These results suggest that swimming stress facilitates ACTH release, at least in part via activation of OX-2R.

Adrenocorticotropic Hormone↗
PubMed2006

Association between polycystic ovary syndrome and female-to-male transsexuality.

BACKGROUND: The aim of this study is to understand the relationship between polycystic ovary syndrome (PCOS), altered hormonal characteristics and insulin resistance in female-to-male (FTM) transsexual patients. METHODS: We studied 69 Japanese FTM cases, aged 17-47 years, who were seen in the Gender Identity Disorder Clinic of Sapporo Medical University Hospital between December 2003 and May 2006. The subjects had never received hormonal treatment or sex re-assignment surgery. Prior to treatment, they received physical examinations entailing measurement of anthropometric, metabolic and endocrine parameters, after which we compared the values obtained according to the presence or absence of PCOS and/or obesity. Insulin resistance was determined using the homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: Of the 69 participating FTM cases, 40 (58.0%) were found to have PCOS. Of the 49 for whom HOMA-IR was calculated, 15 (30.6%) also showed insulin resistance, whereas of the 59 for whom adiponectin was measured, 18 (30.5%) showed hypoadiponectinaemia. Of 69 for whom androgens were measured, 29 (39.1%) showed hyperandrogenaemia. Insulin resistance was associated with obesity but not with PCOS. In contrast, hyperandrogenaemia was associated with both PCOS and obesity. CONCLUSION: FTM transsexual patients have a high prevalence of PCOS and hyperandrogenaemia.

Adiponectin↗
PubMed2006

Possible inhibitory role of prolactin-releasing peptide for ACTH release associated with running stress.

Exercise around the lactate threshold induces a stress response, defined as "running stress." We have previously demonstrated that running stress is associated with activation of certain regions of the brain, e.g., the paraventricular hypothalamic nucleus (PVN) and supraoptic nucleus, that are hypothesized to play an integral role in regulating stress-related responses, including ACTH release during running. Thus we investigated the role of prolactin-releasing peptide (PrRP), found in the ventrolateral medulla and the nucleus of the solitary tract, which is known to project to the PVN during running-induced ACTH release. Accumulation of c-Fos in PrRP neurons correlated with running speeds, reaching maximal levels under running stress. Intracerebroventricular injection of neutralizing anti-PrRP antibodies led to increased plasma ACTH level and blood lactate accumulation during running stress, but not during restraint stress. Exogenous intracerebroventricular administration of low doses of PrRP had the opposite effects. Therefore, our results suggest that, during running stress, PrRP-containing neurons are activated in an exercise intensity-dependent manner, and likewise the produced endogenous PrRP attenuates ACTH release and blood lactate accumulation during running stress. Here we provide a novel perspective on understanding of PrRP in the endocrine-metabolic response associated with running stress.

Adrenocorticotropic Hormone↗
PubMed2006

Human pulmonary surfactant protein D binds the extracellular domains of Toll-like receptors 2 and 4 through the carbohydrate recognition domain by a mechanism different from its binding to phosphatidylinositol and lipopolysaccharide.

Pulmonary surfactant protein D (SP-D), a member of the collectin group of innate immune proteins, plays important roles in lipopolysaccharide (LPS) recognition. We have previously shown that surfactant protein A (SP-A), a homologous collectin, interacts with Toll-like receptor (TLR) 2, resulting in alteration of TLR2-mediated signaling. In this study, we found that natural and recombinant SP-Ds exhibited specific binding to the extracellular domains of soluble forms of recombinant TLR2 (sTLR2) and TLR4 (sTLR4). Binding was concentration- and Ca2+-dependent, and SP-D bound to N-glycosidase F-treated sTLRs on ligand blots. Anti-SP-D monoclonal antibody 7A10 blocked binding of SP-D to sTLR2 and sTLR4, but there was no inhibitory effect of monoclonal 7C6. Epitope mapping with recombinant proteins consisting of the carbohydrate recognition domain (CRD) and the neck domain plus CRD (NCRD) localized binding sites for 7A10 and 7C6 to sequential epitopes associated with the CRD and the neck domain, respectively. Interactions with 7A10 but not 7C6 were blocked by prior binding of the NCRD to sTLRs. Although antibody 7A10 significantly inhibited the binding of SP-D to its major surfactant-associated ligand, phosphatidylinositol (PI), and Escherichia coli Rc LPS, 7C6 enhanced binding to both molecules. An SP-D(E321Q, N323D) mutant with altered carbohydrate specificity exhibited attenuated PI binding but showed an increased level of binding to sTLRs. Thus, human SP-D binds the extracellular domains of TLR2 and TLR4 through its CRD by a mechanism different from its binding to PI and LPS.

Antibodies, Monoclonal↗
PubMed2006

SYT-SSX1 and SYT-SSX2 interfere with repression of E-cadherin by snail and slug: a potential mechanism for aberrant mesenchymal to epithelial transition in human synovial sarcoma.

Synovial sarcoma is a primitive mesenchymal neoplasm characterized in almost all cases by a t(X;18) fusing the SYT transcriptional coactivator gene with either SSX1 or SSX2, with the resulting fusion gene encoding an aberrant transcriptional regulator. A subset of synovial sarcoma, predominantly cases with the SYT-SSX1 fusion, shows foci of morphologic epithelial differentiation in the form of nests of glandular epithelium. The striking spontaneous mesenchymal to epithelial differentiation in this cancer is reminiscent of a developmental switch, but the only clue to its mechanistic basis has been the observation that most cases of synovial sarcoma with glandular epithelial differentiation (GED) contain SYT-SSX1 instead of SYT-SSX2. We report here that SYT-SSX1 and SYT-SSX2 interact preferentially with Snail or Slug, respectively, and prevent these transcriptional repressors from binding to the proximal E-cadherin promoter as shown by coimmunoprecipitation and chromatin immunoprecipitation. Luciferase reporter assays reveal that SYT-SSX1 and SYT-SSX2 can respectively overcome the Snail- or Slug-mediated repression of E-cadherin transcription. This provides a mechanism by which E-cadherin expression, a prerequisite of epithelial differentiation, is aberrantly derepressed in synovial sarcoma and may also explain the association of GED with the SYT-SSX1 fusion because it interferes with Snail, the stronger repressor of the E-cadherin promoter. Thus, our data provide a mechanistic basis for the observed heterogeneity in the acquisition of epithelial characteristics in synovial sarcoma and highlight the potential role of differential interactions with Snail or Slug in modulating this phenotypic transition.

Amino Acid Sequence↗
PubMed2006

Coordinated elevation of membrane type 1-matrix metalloproteinase and matrix metalloproteinase-2 expression in rat uterus during postpartum involution.

BACKGROUND: The changes occurring in the rodent uterus after parturition can be used as a model of extensive tissue remodeling. As the uterus returns to its prepregnancy state, the involuting uterus undergoes a rapid reduction in size primarily due to the degradation of the extracellular matrix, particularly collagen. Membrane type-I matrix metalloproteinase (MT1-MMP) is one of the major proteinases that degrades collagen and is the most abundant MMP form in the uterus. Matrix metalloproteinase-2(MMP-2) can degrade type I collagen, although its main function is to degrade type IV collagen found in the basement membrane. To understand the expression patterns of matrix metalloproteinases (MMPs) in the rat uterus, we analyzed their activities in postpartum uterine involution. METHODS: We performed gelatin zymography, northern blot analysis and immunohistochemistry to compare the expression levels of MT1-MMP, MMP-2, matrix metalloproteinase-9 (MMP-9) and the tissue inhibitors of MMPs-1 and 2 (TIMP-1 and TIMP-2) in the rat uterus 18 h, 36 h and 5 days after parturition with their expression levels during pregnancy (day 20). RESULTS: We found that both MT1-MMP and MMP-2 localized mainly in the cytoplasm of uterine interstitial cells. The expression levels of MT1-MMP and MMP-2 mRNAs and the catalytic activities of the expressed proteins significantly increased 18 h and 36 h after parturition, but at postpartum day 5, their mRNA expression levels and catalytic activities decreased markedly. The expression levels of MMP-9 increased 18 h and 36 h after parturition as determined by gelatin zymography including the expression levels of TIMP-1 and TIMP-2. CONCLUSION: These expression patterns indicate that MT1-MMP, MMP-2, MMP-9, TIMP-1 and TIMP-2 may play key roles in uterine postpartum involution and subsequent functional regenerative processes.

Animals↗
PubMed2006

Possible involvement of gap junctions in the barrier function of tight junctions of brain and lung endothelial cells.

Gap-junction plaques are often observed with tight-junction strands of vascular endothelial cells but the molecular interaction and functional relationships between these two junctions remain obscure. We herein show that gap-junction proteins connexin40 (Cx40) and Cx43 are colocalized and coprecipitated with tight-junction molecules occludin, claudin-5, and ZO-1 in porcine blood-brain barrier (BBB) endothelial cells. Gap junction blockers 18beta-glycyrrhetinic acid (18beta-GA) and oleamide (OA) did not influence expression of Cx40, Cx43, occludin, claudin-5, junctional adhesion molecule (JAM)-A, JAM-B, JAM-C, or ZO-1, or their subcellular localization in the porcine BBB endothelial cells. In contrast, these gap-junction blocking agents inhibited the barrier function of tight junctions in cells, determined by measurement of transendothelial electrical resistance and paracellular flux of mannitol and inulin. 18beta-GA also significantly reduced the barrier property in rat lung endothelial (RLE) cells expressing doxycycline-induced claudin-1, but did not change the interaction between Cx43 and either claudin-1 or ZO-1, nor their expression levels or subcellular distribution. These findings suggest that Cx40- and/or Cx43-based gap junctions might be required to maintain the endothelial barrier function without altering the expression and localization of the tight-junction components analyzed.

Animals↗
PubMed2006

Successful delivery after vaginal radical trachelectomy for invasive uterine cervical cancer.

A 32-year-old Japanese woman was diagnosed as having stage Ib1 adenocarcinoma by diagnostic laser conization at a local hospital. She was admitted to our hospital for fertility-sparing treatment. A radical trachelectomy (RT) was performed using the laparoscopic vaginal procedure. The procedure was started with a laparoscopic pelvic lymphadenectomy. As the lymph nodes were tumor free, RT was carried out transvaginally. The excised uterine cervix and lymph nodes were pathologically negative for cancer. Eight months after the operation, the patient became pregnant without any artificial reproduction techniques. At 17 weeks of gestation, she was admitted to our hospital again for a threatened abortion. Continuous tocolytic treatment with ritodrine and daily administration of a granulocyte elastase inhibitor vaginal suppository were given. At 32 weeks of gestation, she underwent emergency cesarean section because of sudden premature rupture of the membranes. A girl weighing 1991 g was delivered, with Apgar scores of 7 and 8 at 1 and 5 min, respectively. Both the mother and the baby were discharged without trouble. This is the first successful case in Japan of delivery after vaginal RT for invasive uterine cervical cancer.

Adult↗
PubMed2006

Altered expression of Fas/Fas ligand/caspase 8 and membrane type 1-matrix metalloproteinase in atretic follicles within dehydroepiandrosterone-induced polycystic ovaries in rats.

One of the characteristics of polycystic ovary syndrome (PCOS) is the presence of cystic follicles in various stages of growth and atresia, the latter of which is known to be the result of apoptosis and tissue remodeling. To further investigate the process of follicular atresia, we compared ovarian expression and localization of Fas, Fas ligand (FasL), casapse-8 and membrane-type1 matrix metalloproteinase (MT1-MMP) in rats treated with dehydroepiandrosterone (DHEA) as a model of PCOS, and in control rats. We found that the numbers of TdT-mediated dUTP-biotin nick end-labeling (TUNEL)-positive follicles were significantly higher in ovaries from PCOS rats than in those from control rats (P < 0.05), as were ovarian levels of FasL mRNA and protein, processed caspase-8 protein and MT1-MMP mRNA. Correspondingly, we also observed an increase in the level of MTI-MMP catalytic activity and a decrease in the level of pro-caspase-8 protein. In addition, immunohistochemical analyses showed that MT1-MMP and FasL co-localize with TUNEL-positive apoptotic granulosa cells within atretic follicles of PCOS ovaries. Our results suggest that under the PCOS-like conditions induced by DHEA, the Fas/FasL/Caspase-8 (death receptor dependent) pathway is pivotal for follicular atresia, and that increased levels of MT1-MMP likely play an important role in tissue remodeling during structural luteolysis.

Animals↗
PubMed2006

Nuclear beta-catenin correlates with cyclin D1 expression in spindle and pleomorphic sarcomas but not in synovial sarcoma.

Nuclear beta-catenin staining in soft tissue sarcomas (STSs) has been shown to correlate with tumor progression as assessed by proliferative activity or poor prognosis. Frequent activation of Wnt signaling pathway has been also shown in synovial sarcoma (SS), suggesting a specific role of this pathway in SS. We examined roles of nuclear beta-catenin staining within soft tissue sarcomas. Immunohistochemical detection of nuclear beta-catenin accumulation correlated with cyclin D1 overexpression in spindle cell and pleomorphic sarcomas (P = .037), and the expression of these proteins evenly distributed throughout each section. In some cases, strong beta-catenin nuclear staining was observed in highly pleomorphic and mitotic cells. Furthermore, tumors with nuclear beta-catenin accumulation showed statistically significant increasing cyclin D1 mRNA expression level compared with those without (P = .023). Cyclin D1 mRNA expression levels were statistically higher in tumors with cyclin D1 overexpression than in tumors without (P = .037), suggesting that cyclin D1 overexpression is due to transcriptional activation. However, these correlations could not be detected in SS. In biphasic SS, beta-catenin nuclear staining was observed in spindle cells, whereas cyclin D1 nuclear staining was seen in glandular areas where beta-catenin kept membranous expression. Mutations in exon 3 of the beta-catenin gene and in the mutation cluster region of adenomatous polyposis coli gene were absent in this series of cases. Thus, cyclin D1 could be considered as one of the targets of the nuclear beta-catenin in spindle cell and pleomorphic sarcomas. A possible association between beta-catenin accumulation and spindle cell morphogenesis may exist in SS.

Cell Nucleus↗
PubMed2006

Efficacy of accelerated partial breast irradiation as a neoadjuvant treatment for patients with breast cancer: a pilot study.

BACKGROUND: Breast conserving treatment (BCT) consists of breast-conserving operation and followed by whole-breast irradiation (WBI). Accelerated partial breast irradiation (APBI) is being considered as a possible alternative to WBI. Neoadjuvant APBI might provide more benefit than postsurgical APBI because tumor downstaging will enhance the likelihood of BCT. METHODS: APBI was delivered as 50 Gy in 5 fractions over 5 days before operation for patients with breast cancer of 3 to 4 cm in diameter. Patients with tumors 3 cm or less were deemed to be candidates for breast-conserving operation. RESULTS: Between September 1998 and August 1999, 12 women were enrolled. The mean tumor diameter and volume were reduced from 3.4 to 1.8 cm (reduction rate: 47%) and from 8.1 to 2.2 cm3 (reduction rate: 71%), respectively. The mean pathologic tumor size was 1.5 cm, and a complete pathologic response was found in 1 patient (8%). All patients were eligible for breast-conserving operation. No ipsilateral breast recurrences have been observed to date. CONCLUSIONS: This is the first report of neoadjuvant APBI for relatively large breast cancers. Although the number of participants was small, these results would encourage the development of clinical trials exploring the efficacy of neoadjuvant APBI.

Adult↗
PubMed2006

DNA hypermethylation status of multiple genes in soft tissue sarcomas.

The aberrant methylation of promoter CpG islands is known to be a major inactivation mechanism of tumor-related genes. To determine the clinicopathological significance of gene promoter methylation in soft tissue sarcomas, we examined the promoter methylation status of 10 tumor-related genes in 65 soft tissue sarcomas and 19 adjacent non-neoplastic tissues by methylation-specific PCR. The methylation frequencies of tumor-related genes tested in soft tissue sarcomas were 17 (26%) for RASSF1A, 11 (17%) for DAP kinase, 10 (15%) for MGMT, nine (14%) for GSTP1, eight (12%) for PTEN, six (9%) for p16 and hMLH1, five (8%) for hMSH2, two (3%) for p14, and one (2%) for RB. Promoter methylation of these genes was not recognized in non-neoplastic tissues. All those cases of soft tissue sarcoma that had MGMT methylation, with the exception of one case of malignant peripheral nerve sheath tumor, showed large tumor size (> or = 10 cm) or recurrence. Moreover, eight of 10 cases with MGMT methylation revealed high American Joint Committee on Cancer stage. Seven of 10 cases (70%) with MGMT methylation showed a loss of MGMT expression by immunohistochemistry. In addition, MGMT methylation status had a statistically significant correlation with a loss of MGMT expression (P=0.014). In conclusion, although methylation of tumor-related genes was a relatively rare event in soft tissue sarcomas, methylation was tumor-specific. Of 10 tumor-related genes, cases with MGMT methylation had a tendency to be aggressive behavior. Moreover, MGMT methylation was closely associated with a loss of MGMT expression. Although our findings need to be extending to a large series, promoter methylation of tumor-related genes is likely to have an association with the pathogenesis of soft tissue sarcomas. Furthermore, MGMT methylation may be associated with tumor aggressiveness and the inactivation of MGMT gene.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Prognostic significance of dysadherin expression in epithelioid sarcoma and its diagnostic utility in distinguishing epithelioid sarcoma from malignant rhabdoid tumor.

Dysadherin is a cancer-associated cell membrane glycoprotein, which downregulates E-cadherin and promotes metastasis. We studied the clinicopathological features in 72 cases of epithelioid sarcoma and in six cases of malignant rhabdoid tumor, and also assessed the immunohistochemical expression of dysadherin, E-cadherin and MIB-1 in epithelioid sarcoma and malignant rhabdoid tumor cases. In addition, we compared dysadherin mRNA expression between epithelioid sarcoma and malignant rhabdoid tumor cell lines, using RT-PCR and real-time quantitative RT-PCR analysis. Immunohistochemical dysadherin expression was more frequently observed in proximal-type epithelioid sarcoma (71%) in comparison with distal-type epithelioid sarcoma (36%) (P = 0.037). Furthermore, seven proximal-type epithelioid sarcoma cases mimicking malignant rhabdoid tumor (histologically classified as the large cell type, accompanied by frequent rhabdoid cells and located in deep soft tissue) were all positive for dysadherin (100%), whereas dysadherin expression was not detected at all in any of the true six malignant rhabdoid tumors (0%). Cell lines established from proximal-type epithelioid sarcoma revealed significantly higher levels of dysadherin mRNA expression, compared with the levels seen in malignant rhabdoid tumor cell lines by real-time quantitative RT-PCR (P = 0.0433). Epithelioid sarcoma patients with dysadherin expression survived for a significantly shorter time than those without dysadherin expression (P = 0.001). In multivariate analysis, dysadherin immunopositivity (P = 0.0004) was one of the two independent adverse prognostic factors. We conclude that dysadherin expression in epithelioid sarcoma is a significant poor prognostic factor and that it is a powerful diagnostic marker for distinguishing epithelioid sarcoma, including the proximal-type epithelioid sarcoma, from malignant rhabdoid tumor. In epithelioid sarcoma, especially in proximal-type epithelioid sarcoma, increased cell disadhesion and motility by dysadherin plays an important role to acquire aggressive biological behavior. However, in malignant rhabdoid tumor, cell growth cycle that is regulated by hSNF5/INI1 gene seems to be critical to lethal biological behavior rather than dysadherin.

Biomarkers, Tumor↗
PubMed2005

Differential responsiveness of c-Fos expression in the rat medulla oblongata to different treadmill running speeds.

Expression of the inducible transcription factor c-Fos was mapped in the rat medulla oblongata to identify the brain areas respond to different running speeds. Rats were subjected to 30 min of running, either at high speed, low speed or just sitting on a treadmill (control). Blood lactate levels were measured to confirm the physiological impact of different exercise intensities. The number of c-Fos-ir cells was counted and their spatial distributions were mapped through the rostral to the caudal level in the medulla. A statistically significant exercise intensity-dependent induction of c-Fos was observed in the nucleus of the solitary tract (NTS) and caudal ventrolateral medulla (CVL) in the medulla. Further, c-Fos induction was more predominant in the caudal part of each nucleus. The present data clearly show that different running speeds cause differential activation of each nucleus in the medulla, and in particular, the caudal parts of the NTS and the CVL are the most responsive to speed changes. The present study identifies brain areas newly found to be responsive to changes in running speed. These findings are likely to be particularly helpful in studies of specific neural circuits and their functions in response to different running speeds.

Animals↗
PubMed2005

Activation of A1 and A2 noradrenergic neurons in response to running in the rat.

Since running accompanied with blood lactate accumulation stimulates the release of adrenocorticotropic hormone (ACTH), running above the lactate threshold (LT) acts as stress (running stress). To examine whether A1/A2 noradrenergic neurons that project to the hypothalamus activate under running stress, c-Fos immunohistochemistry was used to compare the effects of running with or without stress response on A1/A2 noradrenergic neurons. Blood lactate and plasma ACTH concentrations significantly increased in the running stress group, but not in the running without stress response and control groups, confirming different physiological impacts between different intensity of running with or without stress. Running stress markedly increased c-Fos accumulation in the A1/A2 noradrenergic neurons. Running without stress response also induced a significant increase in c-Fos expression in the A1/A2 noradrenergic neurons, and the percentage of the increase was smaller than that of running stress. The extent of c-Fos expression in the A1/A2 noradrenergic neurons correlates with exercise intensity, signifying that this neuronal activation is running speed-dependent. We thus suggest that A1/A2 noradrenergic neurons are activated in response to not only running stress, but also to other physiological running, enhanced by non-stressful running. These findings will be helpful in studies of specific neurocircuits and in identifying their functions in response to running at different intensities.

Adaptation, Physiological↗
PubMed2005

Hyperstimulation and a gonadotropin-releasing hormone agonist modulate ovarian vascular permeability by altering expression of the tight junction protein claudin-5.

We investigated the mechanism by which a GnRH agonist (GnRHa) affects ovarian vascularity, vascular permeability, and expression of the tight junction protein claudin-5 in a rat model of ovarian hyperstimulation syndrome (OHSS). Hyperstimulated rats received excessive doses of pregnant mare serum gonadotropin (PMSG; 50 IU/d) for 4 consecutive days, from d 25 to 28 of life, followed by 25 IU human chorionic gonadotropin (hCG) on d 29. Control rats received 10 IU PMSG on d 27 of life, followed by 10 IU hCG on d 29. GnRHa (leuprolide 100 microg/kg.d) was administered to some hyperstimulated rats either on d 29 and 30 (short-term GnRHa treatment) or from d 25 to 30 (long-term GnRHa treatment). Ovarian vascular density (vessels per 10 mm(2)) and vessel endothelial area (percent) were assessed by immunohistochemical analysis of the distribution of von Willebrand factor, whereas vascular permeability was evaluated based on leakage of Evans blue. High doses of PMSG and hCG significantly increased ovarian weight, vascular permeability, vascular density, and the vessel endothelial area and significantly reduced expression of claudin-5 protein and mRNA. All of these effects were significantly and dose-dependently inhibited by administration of GnRHa. This suggests that reduced expression of claudin-5 plays a crucial role in the increased ovarian vascular permeability seen in OHSS and that its expression can be modulated by GnRHa treatment. Indeed, preventing redistribution of tight junction proteins in endothelial cells and the resultant loss of endothelial barrier architecture might be the key to protecting patients against massive extravascular fluid accumulation in cases of OHSS.

Analysis of Variance↗
PubMed2005

Behavior of tight-junction, adherens-junction and cell polarity proteins during HNF-4alpha-induced epithelial polarization.

We previously reported that expression of tight-junction molecules occludin, claudin-6 and claudin-7, as well as establishment of epithelial polarity, was triggered in mouse F9 cells expressing hepatocyte nuclear factor (HNF)-4alpha [H. Chiba, T. Gotoh, T. Kojima, S. Satohisa, K. Kikuchi, M. Osanai, N. Sawada. Hepatocyte nuclear factor (HNF)-4alpha triggers formation of functional tight junctions and establishment of polarized epithelial morphology in F9 embryonal carcinoma cells, Exp. Cell Res. 286 (2003) 288-297]. Using these cells, we examined in the present study behavior of tight-junction, adherens-junction and cell polarity proteins and elucidated the molecular mechanism behind HNF-4alpha-initiated junction formation and epithelial polarization. We herein show that not only ZO-1 and ZO-2, but also ZO-3, junctional adhesion molecule (JAM)-B, JAM-C and cell polarity proteins PAR-3, PAR-6 and atypical protein kinase C (aPKC) accumulate at primordial adherens junctions in undifferentiated F9 cells. In contrast, CRB3, Pals1 and PATJ appeared to exhibit distinct subcellular localization in immature cells. Induced expression of HNF-4alpha led to translocation of these tight-junction and cell polarity proteins to beltlike tight junctions, where occludin, claudin-6 and claudin-7 were assembled, in differentiated cells. Interestingly, PAR-6, aPKC, CRB3 and Pals1, but not PAR-3 or PATJ, were also concentrated on the apical membranes in differentiated cells. These findings indicate that HNF-4alpha provokes not only expression of tight-junction adhesion molecules, but also modulation of subcellular distribution of junction and cell polarity proteins, resulting in junction formation and epithelial polarization.

Adherens Junctions↗