ASPP2 suppression promotes malignancy via LSR and YAP in human endometrial cancer.

Apoptosis-stimulating p53 protein 2 (ASPP2) is an apoptosis inducer that acts via binding with p53 and epithelial polarity molecule PAR3. Lipolysis-stimulated lipoprotein receptor (LSR) is an important molecule at tricellular contacts, and loss of LSR promotes cell migration and invasion via Yes-associated protein (YAP) in human endometrial cancer cells. In the present study, to find how ASPP2 suppression promotes malignancy in human endometrial cancer, we investigated its mechanisms including the relationship with LSR. In endometriosis and endometrial cancers (G1 and G2), ASPP2 was observed as well as PAR3 and LSR in the subapical region. ASPP2 decreased in G3 endometrial cancer compared to G1. In human endometrial cancer cell line Sawano, ASPP2 was colocalized with LSR and tricellulin at tricellular contacts and binding to PAR3, LSR, and tricellulin in the confluent state. ASPP2 suppression promoted cell migration and invasion, decreased LSR expression, and induced expression of phosphorylated YAP, claudin-1, -4, and -7 as effectively as the loss of LSR. Knockdown of YAP prevented the upregulation of pYAP, cell migration and invasion induced by the ASPP2 suppression. Treatment with a specific antibody against ASPP2 downregulated ASPP2 and LSR, affected F-actin at tricellular contacts, upregulated expression of pYAP and claudin-1, and induced cell migration and invasion via YAP. In normal human endometrial epithelial cells, ASPP2 was in part colocalized with LSR at tricellular contacts and knockdown of ASPP2 or LSR induced expression of claudin-1 and claudin-4. ASPP2 suppression promoted cell invasion and migration via LSR and YAP in human endometrial cancer cells.

Efficacy of liquid-based genetic diagnosis of endometrial cancer.

Although liquid-based cytology (LBC) has increased the sensitivity of cytological diagnosis of endometrial cancer (EC) compared with conventional smear cytology, the sensitivity of LBC for the detection of EC is between 70% and 96% and remains unsatisfactory. In the present study, we compared the efficacy of LBC with liquid-based genetic diagnosis (LBGDx) by amplicon sequencing of five genes including PTEN, PIK3CA, CTNNB1, KRAS, and TP53 in 48 LBC subjects who underwent endometrial screening. Consequently, LBC classified 15 samples as "positive or suspicious for malignancy" and the 15 were later confirmed as EC. However, LBC failed to identify five cases who were diagnosed as EC by additional transvaginal ultrasound and endometrial curettage, indicating that the sensitivity of cytology alone was 75% (15/20). LBGDx identified 11 pathogenic PTEN variants in 10 subjects, six PIK3CA variants in nine, three CTNNB1 variants in five, two KRAS variants in four, and three TP53 variants in three. Collectively, at least one pathogenic variant was identified in 19 subjects, which included 17 EC (15 endometrioid carcinoma and 2 endometrial carcinosarcomas), and one cervical adenocarcinoma. However, LBGDx did not identify any pathogenic mutations in three of the 20 EC, indicating that the sensitivity of LBGDx alone was 85% (17/20). Although five EC were negative for malignancy by LBC and three were negative for pathogenic mutations by LBGDx, the combination of LBC and LBGDx would successfully diagnose all 20 EC. These data suggested that LBGDx is a useful strategy to improve the sensitivity of screening of EC by LBC.

Rap1GAP inhibits tumor progression in endometrial cancer.

OBJECTIVE: Endometrioid adenocarcinoma (EAC) is a common endometrial cancer with recent dramatic increases in incidence. Previous findings indicate that Rap1GAP acts as a tumor suppressor inhibiting Ras superfamily protein Rap1 in multiple aggressive carcinomas; however, Rap1GAP expression in EAC has not been investigated. In this study, the tumor suppressing activity of Rap1GAP in EAC was explored. METHODS: EAC cell lines were used to examine Rap1GAP levels by real-time RT-PCR and western blotting and the effects of Rap1GAP on cancer cell invasion and migration. Rap1GAP expression was analyzed by immunohistochemical staining for Rap1GAP, E-cadherin in surgically resected tumors of 114 EAC patients scored according to EAC differentiation grade. Prognostic variables such as age, stage, grade, tumor size, and immunostaining for Rap1GAP, E-cadherin were evaluated using Cox regression multivariate analysis. RESULTS: Low Rap1GAP expression was detected in poorly differentiated EAC cells. Rap1GAP deficiency significantly accelerated while Rap1 deficiency decreased cancer cell migration and invasion. Patients with higher Rap1GAP, E-cadherin, and especially combined Rap1GAP/E-cadherin levels had better overall survival than EAC patients with no or weak expression. In addition, Rap1GAP expression was an independent prognostic factor in EAC. CONCLUSIONS: Inhibition of Rap1GAP expression increases EAC cell migration and invasion through upregulation of Rap1. Low expression of Rap1GAP correlates with poor EAC differentiation. Our findings suggest that Rap1GAP is an important tumor suppressor with high prognostic value in EAC.

Palonosetron in combination with 1-day versus 3-day dexamethasone for prevention of nausea and vomiting following paclitaxel and carboplatin in patients with gynecologic cancers: A randomized, multicenter, phase-II trial.

AIM: Chemotherapy-induced nausea and vomiting (CINV) can affect a patient's quality of life, leading to poor compliance with further treatments. Previous studies have provided minimal data about carboplatin-based regimens. Female sex is a known risk factor for CINV. The purpose of this study was to evaluate palonosetron plus single-dose dexamethasone (DEX) for preventing CINV caused by carboplatin plus paclitaxel combination therapy (TC regimen) in patients with gynecologic cancers. MATERIAL AND METHODS: Patients were recruited for this phase-II, multicenter, randomized trial from 12 hospitals in Hokkaido, Japan. Eligible patients were women with uterine cervical, endometrial or ovarian cancer scheduled to receive conventional TC regimen or dose-dense TC regimen; 116 patients were randomly assigned to receive palonosetron in combination with 1-day DEX or 3-day DEX. RESULTS: During the overall period, complete response (CR) was observed in 67.9% (95% confidence interval, 53.7-80.1) of patients in the 3-day DEX arm, and 60.7% (95% confidence interval, 46.8-73.5) of patients in the 1-day DEX arm; CR was significantly lower in the 1-day DEX arm if motion sickness was already present (P = 0.0370). In the severe hyperemesis gravidarum cohort, CR in the 1-day DEX arm tended to be lower than in the 3-day DEX arm. CONCLUSION: Combination therapy of palonosetron and 1-day DEX was effective for subjects undergoing a TC regimen for gynecologic cancers. However, the possibility of reduced efficacy of 1-day only DEX therapy in women undergoing a TC regimen could not be refuted and requires further investigation.

Low-dose SN-38 with paclitaxel induces lethality in human uterine cervical adenocarcinoma cells by increasing caspase activity.

Combination of anticancer drugs may provide a rational molecular basis for novel chemotherapeutic strategies. Paclitaxel and SN-38 (an active metabolite of CPT-11) are effective for many kinds of cancer. Therefore, we investigated the possibility that combination of these drugs could be effective against cervical adenocarcinoma cells. In this study, we examined cell growth inhibition after 96 h using the MTT assay and examined the release of fragmented DNA into the cytoplasm during apoptotic cell death by PI staining. Single and combined use of paclitaxel and SN-38 produced significant cytolethality against the cervical adenocarcinoma cell line CAC-1. Addition of a low concentration of SN-38 reduced the IC50 value of paclitaxel compared to that without SN-38, although the low concentration of paclitaxel did not enhance the cytotoxicity of SN-38. FACS scan analysis suggested that these drugs induced apoptosis and cell cycle arrest, and that caspase-3 and -7 were activated in the process. MTT assay and the IC50 demonstrated that paclitaxel had strong cytotoxicity against CAC-1 as well as other cancer cells. In this study, though only a single cell line was used for the experiment and the data are limited, our results suggest that paclitaxel together with low-dose CPT-11 is a promising basis for a new combination cancer chemotherapy.

Possible involvement of tight junctions, extracellular matrix and nuclear receptors in epithelial differentiation.

Tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane. They consist of four kinds of transmembrane proteins (occludin, claudins, junctional adhesion molecules, and tricellulin) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function. The mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases. In this paper, we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions, extracellular matrix, and nuclear receptors in epithelial differentiation.

ERα increases endometrial cancer cell resistance to cisplatin via upregulation of BAG3.

Endometrial cancer is a leading cause of cancer-associated mortality in women and has a poor prognosis in advanced stages. Our previous study revealed that BCL-2-associated athanogene 3 (BAG3) may contribute to enhancing cell viability through downregulation of microRNA (miR)-29b in endometrial cancer cell lines. In addition, a relationship between estrogen receptor α (ERα) and BAG3 was recently reported in several cancer cell types. The present study investigated the relationship between ERα and BAG3 in endometrial cancer cell lines. The results demonstrated that exogenous ERα overexpression enhanced BAG3 expression in the EMTOKA endometrial cancer cell line, which does not endogenously express ERα, but had no effect on BAG3 expression levels in the Ishikawa cell line, which does endogenously express ERα. In addition, ERα overexpression suppressed miR-29b expression and enhanced the expression of Mcl-1, a mediator situated downstream of BAG3, in EMTOKA cells, but not Ishikawa cells. ERα overexpression also enhanced EMTOKA, but not Ishikawa, endometrial cancer cell viability in the presence of cisplatin. These findings suggested that ERα may contribute to enhancing endometrial cancer cell resistance to anticancer agents through BAG3 overexpression.

Possibility of Targeting Claudin-2 in Therapy for Human Endometrioid Endometrial Carcinoma.

Claudin-2 (CLDN-2) is a leaky-type tight junction protein, and its overexpression increases tumorigenesis of some types of cancer cells. In the present study, to examine the possibility of targeting CLDN-2 in the therapy for endometrioid endometrial adenocarcinoma, we investigated the regulation and role of CLDN-2 in endometriosis and endometrioid endometrial adenocarcinoma. In endometrioid endometrial adenocarcinoma tissues, marked upregulation of CLDN-2 was observed together with malignancy, while in endometriosis tissues, a change in the localization of CLDN-2 was observed. In cells of the endometrial adenocarcinoma cell line Sawano, which highly express CLDN-2, downregulation of CLDN-2 induced by the siRNA upregulated the epithelial barrier and inhibited cell migration. Furthermore, the downregulation of CLDN-2 affected the cell cycle and inhibited cell proliferation. In Sawano cells cultured with high-glucose medium, CLDN-2 expression was downregulated at the mRNA and protein levels. The high-glucose medium upregulated the epithelial barrier, cell proliferation, and migration, and inhibited cell invasion. The histone deacetylase (HDAC) inhibitor tricostatin A (TSA), which has antitumor effects, downregulated CLDN-2 expression, cell proliferation, invasion, and migration, and upregulated the epithelial barrier. The mitochondrial respiration level, an indicator of cancer metabolism, was downregulated by CLDN-2 knockdown and upregulated by the high-glucose condition. Taken together, these results indicated that overexpression of CLDN-2 closely contributed to the malignancy of endometrioid endometrial adenocarcinoma. Downregulation of CLDN-2 via the changes of the glucose concentration and treatment with HDAC inhibitors may be important in the therapy for endometrial cancer.

Epithelial barrier dysfunction and cell migration induction via JNK/cofilin/actin by angubindin-1.

Angulin-1/LSR is a tricellular tight junction molecule, that plays an important role in maintaining the epithelial and endothelial barriers. The actin cytoskeleton at tricellular contacts also contributes to the maintenance of the epithelial barrier. Loss of angulin-1/LSR enhances the migration of various cancer cells. Angubindin-1 is a novel binder to angulin-1/LSR and angulin-3. It is a peptide generated from the angulin-1 binding site of Clostridium perfringens iota toxin, which affects the actin cytoskeleton and decreases the epithelial and endothelial barrier functions. However, its regulatory mechanisms are not well understood. To investigate the regulatory mechanisms of the epithelial barrier dysfunction and cell migration induction by angubindin-1, we used human endometrial cancer cell line Sawano, which has high LSR expression and the epithelial barrier function. Angubindin-1 decreased LSR expression and the epithelial barrier function and increased cell migration. It inhibited the recovery of the epithelial barrier function in a Ca-switch model. At tricellular contacts, sinking of the membrane and an increase of actin fibers near the junctions were caused by angubindin-1. It dynamically changed F-actin from lines to dot-like structures at tricellular contacts. Angubindin-1 transiently increased the phosphorylation of cofilin and JNK, which are involved in the regulation of the intracellular actin cytoskeleton. Furthermore, knockdown of JNK and the JNK inhibitor SP600125 prevented the decrease of the epithelial barrier function and the increase of cell migration induced by angubindin-1. These findings suggest that angubindin-1 might reversibly regulate the epithelial barrier and cell migration at tricellular contacts via JNK/cofilin/actin cytoskeleton dynamics.

Localization of Tricellular Tight Junction Molecule LSR at Midbody and Centrosome During Cytokinesis in Human Epithelial Cells.

Epithelial integrity and barrier function are maintained during cytokinesis in vertebrate epithelial tissues. The changes in localization and the roles of tricellular tight junction molecule lipolysis-stimulated lipoprotein receptor (LSR) during cytokinesis are not well known, although new tricellular tight junctions form at the flank of the midbody during cytokinesis. In this study, we investigated the changes in localization and the role of LSR at the midbody and centrosome during cytokinesis using human endometrial carcinoma cell line Sawano, comparing the tricellular tight junction molecule tricellulin; bicellular tight junction molecules occludin, claudin-7, zonula occludens-1, and cingulin; and the epithelial polarized related molecules apoptosis-stimulating of p53 protein 2, PAR3, and yes-associated protein. During cytokinesis induced by treatment with taxol, the epithelial barrier was maintained and the tricellular tight junction molecules LSR and tricellulin were concentrated at the flank of the acetylated tubulin-positive midbody and in γ-tubulin-positive centrosomes with the dynein adaptor Hook2, whereas the other molecules were localized there as well. All the molecules disappeared by knockdown using small interfering RNAs. Furthermore, by the knockdown of Hook2, the epithelial barrier was maintained and most of the molecules disappeared from the centrosome. These findings suggest that LSR may play crucial roles not only in barrier function but also in cytokinesis.

Downregulation of lipolysis-stimulated lipoprotein receptor promotes cell invasion via claudin-1-mediated matrix metalloproteinases in human endometrial cancer.

Lipolysis-stimulated lipoprotein receptor (LSR) is a novel molecule present at tricellular contacts which recruits tricellulin (TRIC), a molecular component of tricellular tight junctions (tTJs). LSR and TRIC are colocalized with the bicellular tight junction (bTJ) protein claudin (CLDN)-1-based tight junction strands at tricellular corners. Knockdown of LSR in normal epithelial cells affects tTJ formation and the epithelial barrier function. In cancer cells knockdown of LSR has been demonstrated to increase cell invasion. However, the detailed mechanisms of how the downregulation of LSR enhances cell invasion in cancer remain unclear. In the present study, knockdown of LSR by small interfering RNA (siRNA) in Sawano human endometrial adenocarcinoma cells induced cell invasion. In LSR-knockdown Sawano cells, upregulation of CLDN-1 protein, which contributes to the cell invasion via matrix metalloproteinases (MMPs), was observed compared with the control group by western blotting and immunostaining. Knockdown of LSR significantly induced Sp1 transcription factor activity in the CLDN-1 promoter region. In LSR-knockdown Sawano cells, DNA microarray analysis demonstrated that MMP-1, MMP-2 and MMP-10 mRNA levels were increased, and the protein levels of membrane-type 1-MMP, MMP-2, MMP-9 and MMP-10 were shown to be increased on western blots. Knockdown of CLDN-1 with siRNA prevented the upregulation of cell invasion induced by the knockdown of LSR in Sawano cells. On the invasive front of human endometrial carcinoma tissue samples, a decrease in LSR and increase in CLDN-1 protein levels were observed using immunohistochemical methods. In conclusion, the results indicate that the downregulation of LSR promotes cell invasion of human endometrial cancer via CLDN-1 mediation of MMPs. This mechanism is important for studying the association of tTJs with the cellular invasion of cancer.

Loss of tricellular tight junction protein LSR promotes cell invasion and migration via upregulation of TEAD1/AREG in human endometrial cancer.

Lipolysis-stimulated lipoprotein receptor (LSR) is a unique molecule of tricellular contacts of normal and cancer cells. We investigated how the loss of LSR induced cell migration, invasion and proliferation in endometrial cancer cell line Sawano. mRNAs of amphiregulin (AREG) and TEA domain family member 1 (TEAD1) were markedly upregulated by siRNA-LSR. In endometrial cancer tissues, downregulation of LSR and upregulation of AREG were observed together with malignancy, and Yes-associated protein (YAP) was present in the nuclei. siRNA-AREG prevented the cell migration and invasion induced by siRNA-LSR, whereas treatment with AREG induced cell migration and invasion. LSR was colocalized with TRIC, angiomotin (AMOT), Merlin and phosphorylated YAP (pYAP). siRNA-LSR increased expression of pYAP and decreased that of AMOT and Merlin. siRNA-YAP prevented expression of the mRNAs of AREG and TEAD1, and the cell migration and invasion induced by siRNA-LSR. Treatment with dobutamine and 2-deoxy-D-glucose and glucose starvation induced the pYAP expression and prevented the cell migration and invasion induced by siRNA-LSR. siRNA-AMOT decreased the Merlin expression and prevented the cell migration and invasion induced by siRNA-LSR. The loss of LSR promoted cell invasion and migration via upregulation of TEAD1/AREG dependent on YAP/pYAP and AMOT/Merlin in human endometrial cancer cells.

BAG3-mediated Mcl-1 stabilization contributes to drug resistance via interaction with USP9X in ovarian cancer.

Paclitaxel in combination with carboplatin improves survival among patients with susceptible ovarian cancers, but no strategy has been established against resistant ovarian cancers. BAG3 (Bcl-2-associated athanogene 3) is one of six BAG family proteins, which are involved in such cellular processes as proliferation, migration and apoptosis. In addition, expression of BAG3 with Mcl-1, a Bcl-2 family protein, reportedly associates with resistance to chemotherapy. Our aim in this study was to evaluate the functional role of BAG3 and Mcl-1 in ovarian cancer chemoresistance and explore possible new targets for treatment. We found that combined expression of BAG3 and Mcl-1 was significantly associated with a poor prognosis in ovarian cancer patients. In vitro, BAG3 knockdown in ES2 clear ovarian cancer cells significantly increased the efficacy of paclitaxel in combination with the Mcl-1 antagonist MIM1, with or without the Bcl-2 family antagonist ABT737. Moreover, BAG3 was found to positively regulate Mcl-1 levels by binding to and inhibiting USP9X. Our data show that BAG3 and Mcl-1 are key mediators of resistance to chemotherapy in ovarian cancer. In BAG3 knockdown ES2 clear ovarian cancer cells, combination with ABT737 and MIM1 enhanced the efficacy of paclitaxel. These results suggest that inhibiting BAG3 in addition to anti-apoptotic Bcl-2 family proteins may be a useful therapeutic strategy for the treatment of chemoresistant ovarian cancers.

The roles of tricellular tight junction protein lipolysis-stimulated lipoprotein receptor in malignancy of human endometrial cancer cells.

Lipolysis-stimulated lipoprotein receptor (LSR) has been identified as a novel molecular constituent of tricellular contacts that have a barrier function for the cellular sheet. LSR recruits tricellulin (TRIC), which is the first molecular component of tricellular tight junctions. Knockdown of LSR increases cell motility and invasion of certain cancer cells. However, the behavior and the roles of LSR in endometrial cancer remain unknown. In the present study, we investigated the behavior and roles of LSR in normal and endometrial cancer cells in vivo and in vitro. In endometriosis and endometrial cancer, LSR was observed not only in the subapical region but also throughout the lateral region as well as in normal endometrial epithelial cells in the secretory phase, and LSR in the cancer was reduced in correlation with the malignancy. Knockdown of LSR by the siRNA in cells of the endometrial cancer cell line Sawano, induced cell migration, invasion and proliferation, while TRIC relocalized from the tricellular region to the bicellular region at the membrane. In Sawano cells and normal HEEs, a decrease of LSR induced by leptin and an increase of LSR induced by adiponectin and the drugs for type 2 diabetes metformin and berberine were observed via distinct signaling pathways including JAK2/STAT. In Sawano cells, metformin and berberine prevented cell migration and invasion induced by downregulation of LSR by the siRNA and leptin treatment. The dissection of the mechanism in the downregulation of endometrial LSR during obesity is important in developing new diagnostic and therapy for endometrial cancer.

Hypermethylation in promoter region of retinoic acid receptor-beta gene and immunohistochemical findings on retinoic acid receptors in carcinogenesis of endometrium.

In the present study, we analyzed the immunohistochemical findings for the RA receptor (RAR), retinoic X receptor (RXR) and hypermethylation of promoter-region CpG island methylation of RAR-beta2. Immunohistochemistry indicated that though RXR-alpha and -gamma were present in endometrial hyperplasia and cancer, other retinoid receptors were only weakly detected. The hypermethylation of RAR-beta2 was found in 75.0% of endometrial hyperplasia samples and 92.2% of carcinomas. No normal endometria had methylation. This evidence may point to one of the reasons why endometrial hyperplasia acquires high proliferative capacity without differentiation, and the hypermethylation of RAR-beta2 may occur in the early stage of endometrial carcinogenesis.

BAG3 increases the invasiveness of uterine corpus carcinoma cells by suppressing miR-29b and enhancing MMP2 expression.

Approximately 30% of uterine corpus carcinomas are diagnosed at an advanced stage and have a poor prognosis. Our previous study indicated that BCL2-associated athanogene 3 (BAG3) enhances matrix metalloproteinase-2 (MMP2) expression and binds to MMP2 to positively regulate the process of cell invasion in ovarian cancer cells. Recently, altered miRNA expression patterns were observed in several groups of patients with endometrial cancers. One of the altered miRNAs, miR-29b, reportedly reduces tumor invasiveness by suppressing MMP2 expression. Our aim in the present study was to examine the relationships among BAG3, miR-29b and MMP2 in endometrioid adenocarcinoma cells. We found that BAG3 suppresses miR-29b expression and enhances MMP2 expression, which in turn increases cell motility and invasiveness. Moreover, restoration of miR-29b through BAG3 knockdown reduced MMP2 expression, as well as cell motility and invasiveness. Collectively, our findings indicate that BAG3 enhances MMP2 expression by suppressing miR-29b, thereby increasing the metastatic potential of endometrioid adenocarcinomas.

Overexpressed progesterone receptor form B inhibit invasive activity suppressing matrix metalloproteinases in endometrial carcinoma cells.

In this study, we focused on the influence of progesterone and its receptor in invasion and MMPs on endometrial carcinoma cells. The growth of Ishikawa cells, to which an progesterone receptor form B (PR-B) expressing vector was transfected, was inhibited by progesterone as was the inhibition of the expression of cyclin D1. By invasion assay, in conditions with progesterone, the invasiveness of Ishikawa cells was inhibited as well as the expression of (metalloproteinase) MMP-1, -2, -7 and -9 and Ets-1 decreased. These results suggest that activation of PR-B by progesterone results in tumor suppression by inhibiting cell growth and invasiveness via suppression of the expression of MMPs.

Endogenous NO upon estradiol-17ß stimulation and NO donor differentially regulate mitochondrial S-nitrosylation in endothelial cells.

Adduction of a nitric oxide (NO) moiety (NO(•)) to cysteines termed as S-nitrosylation (SNO) has emerged as a crucial mechanism for NO signaling crucial for mediating the vascular effects of estrogens. Mitochondrion is a known vascular risk factor; however, the effects of estrogens on mitochondrial SNO are incompletely understood. In this study we determined the effects of estradiol-17ß (E2ß) on mitochondrial protein SNO in primary human umbilical vein endothelial cells and compared the mitochondrial nitroso-proteomes in E2ß- and a NO donor S-nitrosoglutathione (GSNO)-treated cells using a proteomics approach. Treatment with 10 nM E2ß and 1 mM GSNO for 30 minutes significantly increased the levels of mitochondrial SNO-proteins. Subcellular localization of SNO-proteins showed mitochondria as the major cellular organelle for protein SNO in response to E2ß and GSNO. E2ß stimulated mitochondrial endothelial nitric oxide synthase (eNOS) phosphorylation and mitochondrial protein SNO that was enhanced by overexpression of mitochondrion or Golgi, but not membrane targeting eNOS constructs. We identified 11, 32, and 54 SNO-proteins in the mitochondria from the untreated, E2ß-, and GSNO-treated human umbilical vein endothelial cells, respectively. Comparisons of the nitroso-proteomes revealed that common and different mitochondrial SNO-proteins were affected by endogenous NO on E2ß stimulation and exogenous NO from donor. These SNO-proteins were associated with various mitochondrial functions, including energy and redox regulation, transport, iron homeostasis, translation, mitochondrial morphology, and apoptosis, etc. Collectively, we conclude that estrogens rapidly stimulate protein SNO in endothelial mitochondria via mitochondrial eNOS, providing a mechanism for mediating the vascular effects of estrogens.

The tight-junction protein claudin-6 induces epithelial differentiation from mouse F9 and embryonic stem cells.

During epithelialization, cell adhesions and polarity must be established to maintain tissue assemblies and separate the biological compartments in the body. However, the molecular basis of epithelial morphogenesis, in particular, a role of cell adhesion molecules in epithelial differentiation from stem cells, remains unclear. Here, we show that the stable and conditional expression of a tight-junction protein, claudin-6 (Cldn6), triggers epithelial morphogenesis in mouse F9 stem cells. We also demonstrate that Cldn6 induces the expression of other tight-junction and microvillus molecules including Cldn7, occludin, ZO-1α+, and ezrin/radixin/moesin-binding phosphoprotein50. These events were inhibited by attenuation of Cldn6 using RNA interference or the C-terminal half of Clostridium Perfringens enterotoxin. Furthermore, similar results were obtained in mouse embryonic stem cells. Thus, we have uncovered that the Cldn6 functions as a novel cue to induce epithelial differentiation.

Preeclampsia up-regulates angiogenesis-associated microRNA (i.e., miR-17, -20a, and -20b) that target ephrin-B2 and EPHB4 in human placenta.

CONTEXT: Placental angiogenesis contributes to the pathogenesis of preeclampsia (PE) that affects 5-8% of all human pregnancies. MicroRNA (miRNA) are a class of noncoding 21- to 25-nucleotide RNA that negatively regulate gene expression posttranscriptionly. OBJECTIVE: The aim of this study was to test the hypothesis that miRNA are differentially expressed in healthy term and PE placentas and a subclass of angiogenesis-associated miRNA are increased by PE. DESIGN: Total miRNA were extracted from villous placental tissues from healthy term and severe preeclamptic pregnancies. Differential miRNA expression was analyzed by microarray and real-time quantitative PCR. Angiogenesis-associated miRNA were analyzed by target prediction databases. In situ hybridization was used to localize miRNA. Target verification was performed by transfection of miRNA precursors or antagomirs into endothelial and BeWo cells and luciferase reporter assays. RESULTS: Three highly expressed miRNA (miR-17, -20a, and -20b) were found significantly increased in PE compared with healthy term placentas (n = 10 per group). They target on the same group of genes important for angiogenesis. miR-20b was expressed primarily in villous syncytiotrophoblasts in term placenta. Overexpression or inhibition of miR-20b differentially regulated mRNA expression of those genes in endothelial vs. trophoblast cells. Luciferase reporter assay showed that miR-20b targets EPHB4 and ephrin-B2 that have been shown to be critical for early human placental development. Placental ephrin-B2 mRNA was significantly down-regulated in PE compared with normotensive pregnancies. CONCLUSION: miR-17, miR-20a, and miR-20b are differentially regulated in human placentas by PE. They regulate EPHB4 and ephrin-B2 expression in trophoblast and endothelial cells via the same "seed" sequence, suggesting their roles in early placental development.