The expression of these cell adhesion molecules was detected in 90 osteosarcoma and 20 osteochondroma specimens using immunohistochemistry. Associations between these parameters and clinicopathological data were also examined. The expression rates of CD44V6, CDH11, and β-catenin were 25.0% (5/20), 70.0% (14/20), and 20.0% (4/20) in osteochondroma specimens, respectively. Compared to osteochondromas, the proportions of expression of CD44V6 and β-catenin in osteosarcoma specimens increased to 65.6% (59/90) and 60.0% (54/90), respectively. However, the expression rate of CDH11 in osteosarcomas was reduced to 40.0% (36/90). The expression of these markers was significantly associated with metastasis and overall survival (P < 0.05). Survival analysis revealed that patients with increased expression of CD44V6 and β-catenin as well as decreased expression of CDH11 were correlated with a shorter survival time. Multivariate analysis indicated that clinical stage, metastasis status, and the expression of CD44V6, CDH11, and β-catenin were found to be associated with overall survival. Further, the expression of β-catenin and that of CD44V6 were positively correlated with each other. Thus, our results indicated abnormal expression of CD44V6, CDH11, and β-catenin in osteosarcomas and osteochondromas, which may provide important indicators for further research.This study aimed to examine the expression of and the relationship between CD44V6, CDH11, and  Oste-catenin –9.CD44 is a transmembrane glycoprotein, and some variant isoforms of CD44 (CD44V) are reportedly associated with increased invasion, metastasis, and poor prognosis –12, part2+-dependent hemophilic interactions [Cadherins comprise a major class of cell-cell adhesion molecules that interact with catenins to regulate adhesion and migration by Caractions , 15. Theractions , 17. Cadβ-catenin pathway has been implicated in the progression and pathogenesis of many forms of human cancer [β-catenin signaling plays a role in osteosarcoma development. Several studies have reported that both nuclear and cytoplasmic β-catenin could be detected in osteosarcomas [The Wnt/n cancer . Howeversarcomas  or corresarcomas .β-catenin belong to the family of cell adhesion molecules (CAM), which have been implicated in all aspects of cell growth, migration, and differentiation in vertebrate cells. Much research is focused on the relationship between CAM and cancer [β-catenin in osteosarcomas, we report in this study our findings regarding the immunohistochemical expression and clinicopathological features of these biomarkers in human osteosarcomas. We have also analyzed the correlation of CD44V6, CDH11, and β-catenin with each other.CD44V6, CDH11, and d cancer –15, 19. All patients or their guardians provided informed consent for participation in this study. This study was approved by the local research ethics committee . The exclusion criterion was inadequate follow-up data as well as preoperative chemotherapy or radiotherapy. Surgically resected osteosarcoma specimens were collected from 90 patients at Zhongnan Hospital of Wuhan University and Hubei Cancer Hospital, Hubei, China, between January 2000 and March 2008. The specimens were obtained from the primary site from 50 patients with metastases and 40 without metastases. No diagnostic biopsies were selected for this study. A total of 20 osteochondroma specimens were used as controls. Formalin-fixed, paraffin-embedded blocks were retrieved from the Department of Pathology. All the samples were evaluated for diagnosis by 3 experienced pathologists. Of the 90 patients, 53 were male and 37 were female. The median age of the patients was 18.3 years, with a range of 8–58 years. Clinical stage was graded according to the standard of Enneking et al. [μm) obtained from representative tissue sample blocks were heated at 60°C for 20 min, deparaffinized with xylene, and rehydrated using graded ethanol. After quenching endogenous peroxidase activity with 3% hydrogen peroxide in methanol for 10 min, the slides were microwaved in 10 mmol/L sodium citrate (pH 6.0) for 15 min to retrieve the antigen. The elimination of nonspecific staining was performed with diluted normal goat serum. The sections were then incubated at 37°C for 1 h with primary antibodies against CD44V6 , CDH11 , and β-catenin . Following three successive rinses with phosphate buffered saline (PBS), they were further incubated with secondary antibody  for 40 min at 37°C. The sections were allowed to develop using a DAB color kit  for 5 min and then were counterstained with hematoxylin. For each protein, the immunostaining in patient samples was compared with osteochondroma as a normal control, while negative controls were obtained by omitting the primary antibody, substituted by PBS. The tissues were fixed in 10% buffered formalin and processed using standard procedures. The sections  was calculated based on previously published research . Immunorβ-catenin was assessed by Spearman's correlation analysis. A P value of less than 0.05 was considered statistically significant for all tests.The whole statistical analysis was performed with SPSS version 18.0 software . The correlation between antigen expression and the clinicopathological parameters was assessed by chi-square test or Kruskal-Wallis test when chi-square test was not suitable. Survival rates were estimated by Kaplan-Meier statistics and survival curves were compared by using the Log-rank test. A Cox regression method was used for multivariate analysis. In this paper, we only investigated the relation between expressions of the three markers with overall survival; the metastasis-free survival was not studied. Survival was calculated from the time of the primary operation. An observation was censored at the last followup if the patient was alive or had died of a cause other than osteosarcoma. The correlation between CD44V6, CDH11, and β-catenin in 4 cases (20.0%) (β-catenin in osteosarcoma cases were increased to 65.6% (59/90) and 60.0% (54/90), respectively. However, the rate of expression of CDH11 in osteosarcomas was reduced compared to osteochondromas to 40.0% (36/90). Representative staining in osteosarcoma specimens is depicted in P < 0.05, The different expression levels of the three markers in osteosarcoma and osteochondroma are depicted in  (20.0%) . In contβ-catenin protein staining was associated with metastasis (P < 0.05). The expression of CD44V6, CDH11, and β-catenin was not significantly associated with age, gender, tumor site, and histological subtype . The incidence is higher in adolescents (0.8–1.1/100 000/year at age 15–19), where it accounts for >10% of all solid cancers . The devP = 0.02) were significantly associated with poor overall survival in patients with colorectal cancer. The mechanism of CD44V6 promoting the metastasis of cancer may be attributed to its interactions with various components of the extracellular matrix and its involvement in cell adhesion and critical signaling pathways, for example, Ras and Akt [P < 0.02) of patients with laryngeal squamous cell carcinoma. In addition, the association of CD44V6 expression with malignancy and survival could not be confirmed in several studies investigating osteosarcomas as well as other tumors [Our results revealed that CD44V6 was expressed in 25.0% of the collected samples of osteochondroma, whereas, CD44V6 was expressed in 65.6% of osteosarcoma samples. The findings in our study are consistent with previous research demonstrating that the expression of CD44V6 was correlated with metastasis and poor prognosis in patients with osteosarcoma . It was  and Akt , 35. Nak and Akt  suggeste and Akt . These r and Akt  found th and Akt  reportedr tumors –43. Therin vivo metastasis assay. It has been suggested that tumor-promoting inflammation and antitumor immunity coexist at different points along the path of tumor progression [In this study, a significant correlation was found between CDH11 expression and patient survival, which is consistent with a previous study . Severalgression , and a rgression . Consequβ-catenin is an intracellular protein with two important cellular functions: The cadherin-bound β-catenin is required for cell adhesion, while nuclear β-catenin transmits extracellular-initiated Wnt signals to the nucleus. β-catenin is involved with differentiation and proliferation of cells in a wide variety of tissues, and the Wnt signaling pathway has emerged as an essential pathway in skeletal development and disease [β-catenin staining [β-catenin. Considering this pivotal role of β-catenin, we also analyzed β-catenin expression in our study. Cytoplasmic immunostaining was observed in most cases of osteosarcoma (74/90), and we observed that the expression of β-catenin was significantly increased in osteosarcomas compared to osteochondromas. Furthermore, low expression of β-catenin was correlated with longer survival time. Several studies have revealed that positive cytoplasmic β-catenin expression was associated with the development of metastasis both in vivo and in vitro [β-catenin is thought to play an important role in oncogenesis of osteosarcomas, the role of Wnt signaling in osteosarcomas remains controversial [β-catenin may interact with other proteins, such as NF-kappa B, during oncogenesis [ disease . Severalstaining , 19, 52.in vitro . Althougoversial , 55, 56.ogenesis . Furtherβ-catenin (P < 0.001). The correlation between CD44V6 and β-catenin was concordant with a previous study, which showed that CD44 overexpression (CD44S and CD44V6) was associated with activation of β-catenin, suggesting CD44 is one of the target genes of β-catenin [We also found that the expression of CD44V6 was significantly positively correlated with that of -catenin . β-catenin could be a potential prognostic indicator, especially conjoint analysis of the three markers. The relevant molecular mechanisms require further investigation. Considering that osteosarcoma is a very rare disease, this emphasizes the need for multi-institutional collaboration to identify and validate new biomarkers. An obvious shortcoming of our study is that immunohistochemistry is, at best, a semiquantitative technique. The results should be considered exploratory and caution should be taken in interpreting data. Moreover, as for other prognostic factors, the functions of these molecular markers may vary depending on the tissue context. These results may not be applicable to other tumor types. Another limitation of our study is that osteochondromas were used as a control due to limited conditions, and therefore the results may not be as convincing as a comparison with normal bone specimens. Further studies using normal bone tissue as controls are needed. Despite these limitations, noteworthy results were acquired. The expression of CD44V6, CDH11, and


Activation of beta-catenin is a hallmark of hepatoblastoma (HB) and appears to play a crucial role in its pathogenesis. While aberrant accumulation of the beta-catenin is a common event in HB, mutations or deletions in CTNNB1 (beta-catenin gene) do not always account for the high frequency of protein expression. In this study we have investigated alternative activation of beta-catenin by HGF/c-Met signaling in a large cohort of 98 HB patients enrolled in the SIOPEL-3 clinical trial.We performed immunohistochemistry, using antibodies to total beta-catenin and tyrosine654-phosphorylated beta-catenin, which is a good surrogate marker of HGF/c-Met activation. CTNNB1 mutation analysis was also carried out on all samples. We also investigated beta-catenin pathway activation in two liver cancer cell lines, HuH-6 and HuH-7.Aberrant beta-catenin expression was seen in the cytoplasm and/or nucleus of 87% of tumour samples. Our results also revealed a large subset of HB, 83%, with cytoplasmic expression of tyrosine654-phosphorylated beta-catenin and 30% showing additional nuclear accumulation. Sequence analysis revealed mutations in 15% of our cohort. Statistical analysis showed an association between nuclear expression of c-Met-activated beta-catenin and wild type CTNNB1 . Analysis of total beta-catenin and Y654-beta-catenin in response to HGF activation in the cell lines, mirrors that observed in our HB tumour cohort.We identified a significant subset of hepatoblastoma patients for whom targeting of the c-Met pathway may be a treatment option and also demonstrate distinct mechanisms of beta-catenin activation in HB. Hepatoblastoma is a rare malignant tumor of the liver that occurs in young infants with a median age at diagnosis of 16 months . While mSeveral distinct histological subtypes of hepatoblastoma exist. These include wholly epithelial tumours, with pure fetal and mixed fetal/embryonal histology; tumours with mixed epithelial and mesenchmyal features; and several types of transitional, small and large cell undifferentiated tumours . This heCTNNB1, the gene for β-catenin [Of several distinct developmentally regulated pathways known to be active in hepatoblastoma, such as IGF2/H19 ,8, Notch-catenin ,15,17-19CTNNB1 gene examined in our study (http://www.sanger.ac.uk/genetics/CGP/cosmic) and result in aberrant accumulation of β-catenin in the cell.In the absence of Wnt activation, β-catenin is phosphorylated at specific N-terminal serine and threonine residues by the APC/Axin/GSK3β protein complex resulting in its ubiquitination and subsequent degradation, thus maintaining tight control of β-catenin levels within normal cells . Wnt ligCTNNB1, β-catenin causes pathological gene activation and promotes hepatocyte proliferation [Canonical Wnt/β-catenin signaling directly alters gene expression and is a key regulator of cell proliferation, differentiation, and apoptosis during normal liver development, so mutation or deletion within the β-catenin gene suggests a crucial role of this pathway in the origins of embryonal liver tumors ,23, that upon ligand binding undergoes tyrosine autophosphorylation and in turn triggers the activation of several pathways controlling epithelial-mesenchymal morphogenesis, angiogenesis and cell-cell adhesion . In the Upon signaling by HGF, c-Met becomes phosphorylated at tyrosine residues Y1234 and Y1235 and in turn tyrosine phosphorylates β-catenin at residues Y654 and Y670, causing its dissociation from c-Met at the cell membrane. Tyrosine phosphorylated β-catenin is protected from serine/threonine phosphorylation and subsequent proteosomal degradation allowing its accumulation in the nucleus where it acts as a TCF/LEF transcription cofactor. Thus, HGF/c-Met related activation of β-catenin occurs independent of the canonical Wnt/β-catenin pathway ,27,32.CTNNB1 mutation activation of β-catenin in a large cohort of 84 patients with hepatoblastoma. This characterisation of β-catenin activation by the c-Met pathway may have clinical relevance because several HGF/c-Met small molecule inhibitors are now in early phase clinical trials.Under the auspices of the International Society of Paediatric Oncology Liver Tumour strategy group (SIOPEL) we have investigated the status of β-catenin activation in tumours from patients prospectively enrolled in the SIOPEL 3 hepatoblastoma clinical trial . Here weSIOPEL Liver tumor clinical trials are international, prospective, clinical trials run under the auspices of the SIOP Liver Tumor Strategy Group (SIOPEL). Our cohort comprises patients prospectively enrolled into the SIOPEL 3 clinical trial, a randomised study which opened in March 1998, designed to evaluate the effectiveness of preoperative chemotherapy for standard risk (SR) HB with either cisplatin (CDDP) alone or in combination with doxorubicin (PLADO). A detailed description of the SR patient cohort, its clinical features, staging and outcome has previously been reported . SIOPEL In this study we have accessed a representative cohort of 84 HB patients with clinical, histologic and survival data available for most samples. Both diagnostic and post-chemotherapy samples were available for fourteen patients bringing the total number of samples analysed to 98. In the case of diagnostic samples there was generally just a single formalin-fixed paraffin-embedded (FFPE) tumor block available containing the entire biopsy material on which the diagnosis was made. For each post-chemotherapy case, the most representative FFPE block was identified by examination of slides stained with haematoxylin and eosin (H+E). From the H+E slides, representative tumor and adjacent normal tissue areas were selected by a pathologist (C.M.) for subsequent tissue array construction.A tissue microarray (TMA) was constructed by depositing a 1 mm core of each tumor or normal tissue into a wax recipient block using the Manual Tissue Arrayer I . In cases where tumor heterogeneity was evident, different representative areas of the tumor were sampled for TMA construction. The tissue array block was made in duplicate and 4 μm sections of the TMA blocks were cut for subsequent use in immunohistochemical (IHC) analysis. One TMA section was also stained with H+E for evaluation by pathologists (CM +CT).The sample cohort consists of 98 samples from 84 patients comprising 62 diagnostic tumour biopsies and 36 post-surgical specimens . Histologic information was available for 91 samples. The tumours were examined centrally and classified as either wholly epithelial (n = 33) or mixed epithelial and mesenchymal (n = 54). One tumour was diagnosed as hepatocellular carcinoma (fibrolamellar type) and one as a small cell undifferentiated (SCUD). The epithelial component was further subtyped as pure fetal (n = 43), embryonal (n = 3) or mixed fetal and embryonal (n = 41). Two tumors were subtyped as macrotrabecular type. Focal anaplasia was seen in three tumors and cholangioblastic features in two tumors. Thirteen cases of osteoid formation were noted in the histology reports with additional osteoid formation in a post-chemotherapy sample that lacked osteoid in the diagnostic biopsy. Teratoid features were noted in seven samples.Clinical information that classified patients into the two well-defined risk groups was available for 71 patients in our cohort. Twenty-seven of these were high-risk and forty-four were standard risk. Of these 71 patients, nine were born with low birth weight. PRETEXT classification revealed that there were two PRETEXT stage 1 patients, twenty-two stage 2, thirty-one stage 3 and sixteen stage 4 patients. Only two patients had serum AFP levels of < 100 at diagnosis, making them high-risk. Eight and seven patients had portal vein and vena cava involvement respectively, and extrahepatic intra-abdominal disease was seen in three patients also making them high-risk cases. Metastatic disease was present at diagnosis in thirteen children. Relapse or progression in five HR cases resulted in the death of four patients. In the standard-risk group there were six relapses leading to a single death from disease.Briefly, 4 μm TMA slides were deparaffinized with xylene and ethanol. Antigen retrieval was performed by pressure cooking for 2 minutes in citrate buffer pH6.0. Endogenous peroxidases were blocked with 0.3% hydrogen peroxide and non-specific binding was blocked with normal goat serum. Slides were incubated overnight at 4°C with primary antibodies: Y1234/5-c-Met at 1:300 dilution, Y654-β-catenin at 1:25 dilution and β-catenin at 1:200 . The EnVision HRP/DAB detection system  was used to visualise the results. Slides were lightly counterstained with haematoxylin. All antibodies were optimized for use in IHC using breast tumour control tissue and the appropriate positive and negative controls were used.Immunostaining for β-catenin was scored as normal membranous, diffuse or focal cytoplasmic and diffuse or focal nuclear staining. Staining for Y654-β-catenin was scored as negative, cytoplasmic and/or nuclear staining. Staining for Y1234/5-c-Met was scored as positive (cytoplasmic) or negative. Each array duplicate was also stained and the results collated. The staining intensity was noted but not factored, as differing age of donor blocks and variation in fixation methods can impact on staining intensity. The IHC results were analysed in conjunction with two pathologists (CM and CT).Representative areas of tumour were identified on H+E slides by pathologists and a 1 mm tissue core removed from corresponding areas on paraffin blocks. The RNA was extracted using RecoverALL™ Total Nucleic Acid Isolation kit  as per manufacturer's instructions. Normal adjacent tissue was also removed and RNA extracted where it was available in 62 cases.CTNNB1 gene mutations: Optical density ratio 260/280 of 1.8 - 2.2 and RNA concentration of > 20 ng/ul using a Nanodrop spectrometer . A 150 bp region of the CTNNB1 gene was amplified that includes the β-catenin regulatory region of exon 3 (codons 32-45) using the following primer pair (B-Cat3/B-Cat2): 5' GATTTGATGGAGTTGGACATGG 3' and 5' TCTTCCTCAGGATTGCCTT 3'. Samples were reverse transcribed and amplified using One-Step RT-PCR kit  on a DNA Engine Thermal Cyclar . Reverse transcription was at 50°C for 30 minutes followed by first strand synthesis at 95°C for 15 minutes. 35 cycles of 30 seconds each of denaturation at 94°C, annealing at 52°C and extension at 72°C were carried out. Each reaction contained 1 μl RNA template, 2 μl of enzyme mix, 0.6 mMol of forward and reverse primers, 400 μM of each dNTP, 2.5 mM MgCl2 in a final reaction volume of 50 μl. RT-PCR products were visualised on a 1.5% agarose gel with ethidium bromide. Amplified RT-PCR products were purified using QIAquick PCR purification kit (QIAGEN) as per manufacturer's instructions. Cycle sequencing was carried out on a GeneAmp® PCR System 9700 thermocycler using ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit  using 20 ng RT-PCR product. Sequencing products were run on an ABI 373A sequencer (Applied Biosystems) and all mutations were verified by sequencing the sense and anti-sense strands. Mutation analysis was carried out using Variant™ Reporter Software (Applied Biosystems) and showed good quality traces spanning the region of interest.Samples with the following quality parameters were analysed for Human hepatoblastoma cells, Huh-6  were routinely maintained in minimum essential media (MEM) containing 10% FBS and penicillin/streptomycin. The human hepatocellular carcinoma cell line Huh-7 (JCRB) was cultured in Dulbecco's minimum essential media (D-MEM) with 10% FBS and penicillin/streptomycin. The cells were serum starved for 24 hours prior to treatment with recombinant human HGF  to a concentration of 50 ng/ml for 30, 60, 90 and 120 minutes.®, Missouri, USA). The lysate protein concentrations were determined by bicinchoninic acid protein assay using BSA as a standard . Aliquots of the samples were stored at -80°C until use.Nuclear and cytoplasmic protein fractions were isolated from the cell lines at the timepoints indicated with the CelLytic™ NuCLEAR™ Extraction kit  and Approximately 20 μg of protein sample were run on NuPAGE 4-12% BisTris gels (Invitrogen) with MES-SDS buffer (Invitrogen) using the Xcell SureLock™ Mini-Cell (Invitrogen). The protein marker used was Precision Plus Protein™ Standards (BioRad). The iBlot Gel Transfer Device (Invitrogen) was used for western blotting of proteins. The filters were probed with anti-Y654 β-catenin  and anti-β-catenin . The filters were stripped with a mild stripping buffer containing 1.5% glycine, 0.1% SDS and reprobed after each blot. The immunoblots were incubated for 1 hour with the appropriate secondary antibodies coupled to horseradish peroxidase followed by exposure to ECL plus chemiluminescence reagents  and autoradiography. Immunoblotting with anti-TBP for nuclear proteins and anti-β-actin for cytoplasmic extract was used to confirm equal loading.P-value of < 0.05 was considered statistically significant.Results were analysed with StatView software . Statistical comparisons were made using Pearson's Chi-squared test with Yates' continuity correction data. A CTNNB1 gene varies widely in studies of HB, from 13% to 70% [CTNNB1 mutations in our cohort.We examined total β-catenin protein expression on a HB tissue array using IHC. A total of 87% (85/98) of tumours in our clinical cohort showed aberrant expression of β-catenin in the nucleus and cytoplasm (38/98) or in the cytoplasm alone (47/98) Figure  and 1b. % to 70% ,37. To dCTNNB1 mutations we extracted total RNA from corresponding tissue cores of hepatoblastoma. A 150 pb region of the β-catenin regulatory region of exon 3 of the CTNNB1 gene (codons 32-45) was amplified successfully by RT-PCR in 92 of the samples. Lack of amplification in 6 samples may be due to deletion of exon 3 of CTNNB1. We attempted to amplify a region spanning exon 2 to exon 4 in these 6 samples but were unsuccessful. Therefore our estimation of samples containing deletions may be inaccurate. We identified 11 different point mutations in 14 of 98 samples (15%) (Table CTNNB1 showing that the mutations found were somatic variants (results not shown). The frequency of CTNNB1 mutations (14/98) and possible deletions (6/98) in our cohort was significantly lower than the frequency of aberrant expression of β-catenin protein and statistical analysis shows no correlation between aberrant β-catenin accumulation and gene mutation/deletion. This prompted us to investigate alternative pathways of β-catenin activation in hepatoblastomas in our patient cohort.To identify CTNNB1, 26 (33%) showed nuclear expression of Y654-β-catenin, 44 (56%) showed cytoplasmic staining with only 7 (9%) negative for staining. In contrast, IHC analysis of 20 hepatoblastoma with CTNNB1 mutations or possible deletions showed 5 (25%) were completely negative for Y654-β-catenin . This identified positive staining in the cytoplasm of 82/98 (83%) tumours with an additional 27 (28%) showing nuclear accumulation of Y654-β-catenin. In 78 hepatoblastoma with wild type n Figure , 14 .Statistical analysis shows a significant correlation between nuclear accumulation of tyrosine-phosphorylated β-catenin and HB tumours with wild-type To verify that tyrosine phosphorylation of β-catenin is specifically due to activation of the HGF/c-Met pathway we examined the expression of tyrosine 1234 and 1235-phosphorylated c-Met. These tyrosine residues become auto-phosphorylated specifically in response to HGF ligand binding. Eighty-one tumour samples (82%) were positive for Y1234/5-c-Met staining Figure  and the in vitro total β-catenin and Y654-β-catenin protein expression in response to exposure to HGF in two liver tumour cell lines, one with and one without mutation in CTNNB1 (Huh-6 and Huh-7 respectively). To determine their CTNNB1 status, the Huh-6 and Huh-7 cell lines were analysed for CTNNB1 mutations in exon 3 using RT-PCR and sequencing as outlined above. The hepatoblastoma cell line, Huh-6, carried a missense mutation of G34G > V, a known variant of CTNNB1 while the hepatocellular carcinoma cell line, Huh-7, was wild type CTNNB1  cells due its activating mutation. On exposure to HGF, nuclear and cytoplasmic levels of total β-catenin increased through each timepoint peaking at 90 minutes (Results not shown). In contrast, total β-catenin in the wild type Huh-7 cell line was almost undetectable in the nuclei, and the level seen in the cytoplasm is noticeably lower than that of HuH-6 cells. Upon exposure to HGF, total β-catenin increased in the cytoplasm and was also detected in the nuclei of HuH-7 cells.These cell lines were then routinely cultured and serum starved for 24 hours prior to treatment with HGF at various timepoints. Total β-catenin expression was assessed by immunoblot of the nuclear and cytoplasmic fractions. As expected the Huh-6 cell line bearing a CTNNB1 mutation. No Y654-β-catenin was seen in any untreated cell fraction, in either the wild type or mutant cell lines. However, upon treatment with HGF the wild type Huh-7 cell line showed significantly more β-catenin expression in the nuclei and cytoplasm compared to Huh-6  as activation of ®-catenin through the Wnt pathway and further studies have implicated c-Met activation of ®-catenin in cancer pathogenesis [et al investigated hepatocellular (HCC) tumour characteristics occurring in the presence or absence of mutations in CTNNB1. The authors found that the fibrolamellar (FL) tumours had the highest tyrosine-654-phosphorylated-®-catenin (Y654-®-catenin) levels in the study and these tumours also lacked mutations in the CTNNB1 gene [Danilkovitch-Miagkova ogenesis ,32,39. MNB1 gene .®-catenin protein activation. We used an antibodies to detect tyrosine-654 phosphorylated ®-catenin (Y654-®-catenin) and tyrosine-1234 and 1235-c-Met (Y1234/5-c-Met) as surrogate markers for HGF/c-Met activation. Using this method we found that a large proportion of our cohort (79%) showed c-Met related ®-catenin protein activation. Statistical analysis of tumour groups with and without mutations shows a significant correlation between wild type β-catenin and nuclear accumulation of Y654-β-catenin. This is in keeping with the findings of Cieply et al in hepatocellular carcinoma. To validate our tumour findings, we looked at the effects of HGF treatment on β-catenin and Y654-β-catenin in two liver cancer cell lines, with and without CTNNB1 mutations. The results reflected those seen in HB tumours with c-Met activated β-catenin found only in the cell line with wild type CTNNB1 following HGF treatment. It must be noted, however, that nuclear Y654 β-catenin was seen in two tumours carrying mutations/deletions so an overlap of activation pathways may occur. Furthermore thirteen tumours harbouring mutations/deletions also showed Y654 β-catenin expression in the cytoplasm. Further studies must be carried out to ascertain the effect of mutated β-catenin on the nuclear accumulation of the c-Met related β-catenin pool.This prompted us to analyse our samples for c-Met related CTNNB1 gene nor expression of tyrosine654-phosphorylated β-catenin  that has neither mutations in the Our finding of a large number of tumours (79%) with c-Met activated β-catenin may be relevant to treatment of HB. Although treatment with cisplatin or PLADO followed by resection is highly successful there remains > 15% of HB that suffer from relapse. These relapse patients are often refractive to conventional chemotherapy and have a survival rate of < 20%. The translation of our findings may be important for design of future clinical trials, identifying patients for individual targeted therapy, allowing for fewer side effects or inclusion of c-Met inhibitors in salvage therapy following relapse.®-catenin activation without associated gene mutation. Somatic mutations in exon 3 of the ®-catenin gene have been reported in a variety of cancers . However, aberrant accumulation of ®-catenin without activating mutations has been reported in cancers such as gastrointestinal carcinoid tumour, ovarian cancer, cutaneous lymphoma, malignant melanoma and pancreatic adenocarcinoma [®-catenin may account for the discrepancies between gene mutation and protein expression seen in these tumours and this could indicate susceptibility to RTK-targeting agents in the treatment regimen.Our findings may also have an application in the treatment of other tumours that display arcinoma -46. HGF/The authors declare that they have no competing interests.RP carried out the carried out the immunohistochemistry, the molecular genetic studies, the cell culture and protein work and drafted the manuscript. MC participated in study coordination and sample acquisition. RM carried out statistical analysis and contributed to study design. CM and CT analyzed the immunohistochemistry. AZ carried out the initial histologic examination and diagnosis on the tumours. MS conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Pleurobrachia pileus of 11 orthologues of Wnt signalling genes including all ctenophore Wnt ligands and Fz receptors and several members of the intracellular β-catenin pathway machinery. All genes are strongly expressed around the mouth margin at the oral pole, evoking the Wnt oral centre of cnidarians. This observation is consistent with primary axis polarisation by the Wnts being a universal metazoan feature, secondarily lost in ctenophores during early development but retained in the adult. In addition, local expression of Wnt signalling genes was seen in various anatomical structures of the body including in the locomotory comb rows, where their complex deployment suggests control by the Wnts of local comb polarity. Other important contexts of Wnt involvement which probably evolved before the ctenophore/cnidarian/bilaterian split include proliferating stem cells and progenitors irrespective of cell types, and developing as well as differentiated neuro-sensory structures.Signalling through the Wnt family of secreted proteins originated in a common metazoan ancestor and greatly influenced the evolution of animal body plans. In bilaterians, Wnt signalling plays multiple fundamental roles during embryonic development and in adult tissues, notably in axial patterning, neural development and stem cell regulation. Studies in various cnidarian species have particularly highlighted the evolutionarily conserved role of the Wnt/β-catenin pathway in specification and patterning of the primary embryonic axis. However in another key non-bilaterian phylum, Ctenophora, Wnts are not involved in early establishment of the body axis during embryogenesis. We analysed the expression in the adult of the ctenophore  Amphimedon queenslandicaMnemiopsis leidyiThe Wnts are a metazoan-specific family of extracellularly secreted signal proteins which play central roles in the regulation of cell behaviour and fate, notably during embryonic development and in the control of tissue homeostasis during adult life 2+), Wnts can bind to non-Fz receptors (Ryk) and intracellular effects are triggered by an increase of Ca2+ release to the cytoplasm caused by activation of phospholipase-C (PLC) Wnt ligands classically signal through binding to Frizzled (Fz) membrane receptors, and activated Fz proteins can elicit one or several of distinct Wnt pathways (depending on cellular state) each corresponding to a specific cascade of intracellular events Drosophila), where it plays multiple key roles. These include primary axis establishment during development Wnt/β-catenin is the most intensively studied of the Wnt pathways, particularly in a developmental context in bilaterian models . The Wnt family is nevertheless poorly diversified in ctenophore with only four members. Furthermore, expression of these Wnt genes during embryonic development could not be detected until well after the oral/aboral axis becomes phenotypically apparent, suggesting that unlike in bilaterians, cnidarians and sponges Several recent studies have emphasised the interest of ctenophores , one of Interpretation of these observations in evolutionary terms is problematic because the branching order of the non-bilaterian phyla remains strongly debated. Some phylogenomic analyses have placed ctenophores as the sister group to all other extant metazoans Pleurobrachia pileus is a particularly suited experimental model to investigate the contribution of Wnt signalling to the body plan features typical of this phylum, in particular in the adult, since there is detailed information available concerning anatomy and cellular dynamics of adult tissue renewal for this species. This includes the recent characterisation of multiple localised populations of somatic stem cells The adult ctenophore Pleurobrachia pileus is a marine animal like all ctenophores and lives in the plankton like most other members of the phylum. It displays all characteristic features of the highly original and complex ctenophore body plan, including biradial symmetry  using antisense RNA probes were performed for 11 genes: the four ctenophore Wnt ligands , the two identified paralogues of Dvl, and the P. pileus orthologues of GSK-3β, β-cat and TCF. Among these genes, Wnt ligands, Fz receptors, and Dvl proteins are upstream components shared by several signalling pathways. Fz and Dvl proteins are also thought to function in the so-called Fz-PCP pathway independently from both Wnt ligands and the downstream canonical machinery P. pileus. In this study, we invoke two lines of indirect evidence to identify territories in which Wnts likely activate the β-catenin pathway: (i) synexpression of genes involved in reception of Wnt signal whatever the downstream pathway  together with β-catenin pathway genes ; (ii) more specifically, strong expression of the transcription factor TCF. Indeed TCF is a good marker of Wnt/β-catenin pathway activation  during their reproductive season, between March and June. They were transferred into filtered natural seawater and kept at 16°C. The field work done in the context of this work did not involve any endangered or protected species. Animal collects in the field did not require any specific permission.Adult specimens of P. pileus EST collection publicly available in dbEST (http://www.ncbi.nlm.nih.gov/nucest?term=pleurobrachia). Since we store physical replicates of these ESTs in the lab, antisense RNA probes for these genes were produced directly from the corresponding plasmids. Two of the genes (PpiWnt9 and PpiWntA) were instead recovered from an unpublished Newbler assembly from one run of 454 sequencing of total RNA extracted from mixed embryonic, larval, and adult stages  for 1 hour then washed three times in PBST and dehydrated through a graded series of ethanol and stored in methanol at −20°C. The in situ hybridisation (ISH) protocol was as described in Adults were fixed at 4°C in 4% paraformaldehyde in 50% seawater and 50% PBST . After blocking with 1% bovine serum albumin, samples were incubated with one of the following primary antibodies for 4 hours at room temperature: rat monoclonal anti-tyrosylated α-tubulin , rabbit polyclonal anti-L Dopa , rabbit polyclonal anti-serotonin (5HT) , rabbit polyclonal anti-norepinephrine (NE) , rabbit polyclonal anti-Tyrosine Hydroxylase (TH)  and rabbit polyclonal anti-Vmat2 . A rabbit polyclonal anti-phospho-histone H3 antibody  was used to detect mitotic cells as described in EdU incorporation assays were done using the Click-iT® EdU Alexa Fluor® 488 Imaging Kit from Invitrogen . The protocol was as described in All fluorescence and DIC images were acquired on an Olympus BX61 microscope using a Q-imaging Camera with Image Pro plus software® (Mediacybernetics).2 (7.5%) and seawater (1∶2). A first short fixation (30 sec at 4°C) was done by adding glutaraldehyde in seawater at a final concentration of 3.7%. Then, samples were transferred to a second fixation mix  for 1 hour at 4°C. After several washes in ice cold washing buffer , a post-fixation step was performed in 1% tetraoxyde osmium in washing buffer during 1 hour. Finally, specimens were washed several times in distilled water. Selected animals were dehydrated in a graded series of alcohol then transferred to amylacetate. Following critical-point drying, the specimens were mounted on copper stubs with silver point, coated with gold (330 A layer) in a Polaron sputtering apparatus, and examined on a JEOL JSM 6100 scanning electron microscope .Specimens were relaxed by storing at 4°C in a mix of MgClPleurobrachia pileus are described for 11 genes  which are inferred to function in Wnt signalling based on their orthologies with well-known Wnt signalling genes previously characterised in diverse experimental model species. The phylogenetic analyses establishing these orthology relationships are provided in In this study, expression patterns in the adult body of We describe here in details the expression patterns of these genes in the areas of the body for which we have good morpho-anatomical knowledge allowing interpretation of the patterns in terms of structures, cell types and sometimes cellular dynamics. These are the oral region, the comb rows, the aboral sensory complex  and other epithelial neuro-sensory structures, and the tentacle root. Other expression territories  will not be described here.PpiWnt9 transcripts are restricted to a very narrow oral stripe and within this stripe staining is associated with a subpopulation of cells, more or less scattered along the mouth margin (PpiWntA and PpiWntX are more homogeneously expressed and in a wider stripe (its width of about 4–5 cellular diameters)  , whereash margin ; Fig. 3Dd PpiTcf  are exprf PpiFzB , Fig. 3E cumulate these three expression territories. Furthermore, an additional expression site, two symmetrical transverse spots at the apical organ periphery in the tentacular plane, was seen only for PpiWntX (arrowheads in With the exception of  PpiWnt6  and PpiTPpiWnt9) are strongly expressed in the proximal region of the polar fields  (All genes (except l organ) . For thel organ) , coincidPpiWnt9 are also expressed in the differentiated part of the polar fields , namely within a thin band positioned along the inner border of the polar field marginal zone  is strongly expressed in a longitudinal condensation of the epithelial nerve plexus called the juxta-tentacular nerve cord PpiWnt9 expression in the juxtatentacular nerve cord was seen only in its portion spanning from the apical organ to the tentacular sheath opening . Expression in the juxta-tentacular nerve cord could not be detected for any of the other 10 genes investigated.The Prior to description of gene expression patterns in the comb rows, it will be useful to provide some background about the organisation of the cellular component of combs  and to give some insights about cellular dynamics within them based on original expression patterns of some ciliogenesis genes.The structure of the comb basal cushion, illustrated in external morphology in P. pileus orthologue of the early ciliogenesis marker BBS-5 is expressed at both extremities of the basal cushion  have an expression domain that does not extend to the midline area, with a sharp limit corresponding to the edge of the covering epithelium . In the oral half, a small central spot of higher transcript concentration is observed at least in some combs/individuals for PpiGSK-3β, Ppiβ-cat and PpiTcf  and for some genes on the contrary with marked discrepancy (e.g. β-cat in M. leidyi cydippid expressed everywhere except in the cells forming the comb plates), but with no evidence for an oral Wnt centre.Pang et al.However in the ctenophore M. leidyi embryos and larvae, there is an oral Wnt centre in the adult of the ctenophore P. pileus . Furthermore, Wnt ligands have nested expression domains starting from the oral pole, a situation somewhat reminiscent of nested Wnt expression in cnidarian planula larvae Our expression analyses demonstrate that unlike in . pileus , 3, withP. pileus and M. leidyi? A first scenario would invoke loss of the oral Wnt centre only in the lineage of M. leidyi (which unlike P. pileus belongs to the ctenophore order Lobata). It is not possible at the moment to exclude this hypothesis, in the absence of Wnt expression data in the adult of M. leidyi as well as during embryogenesis in P. pileus. However, a more likely explanation in our view would be that in ctenophores, Wnt/β-catenin signalling has lost its early role in establishment of the oral/aboral axis during embryogenesis, but is nevertheless still involved in its maintenance/patterning in the adult. Consistent with this idea, ctenophore embryogenesis stands apart among non-bilaterians by its strongly mosaic nature and highly stereotyped cleavage pattern How to reconcile these apparently contradictory observations in PpiPiwi1 expression  and areas of stem cell concentration and intense cellular division suggests an ancient role of the Wnt/β-catenin pathway in promoting cell proliferation and possibly other properties of stem cells such as self-renewal and potency. This correlation was observed in the three body areas with localised stem cell populations and spatially ordered cell renewal . Likewise, the circular area around the mouth where all investigated genes are strongly expressed is also characterised by intense cell proliferation and strong pression . Of notePlatynereis dumeriliiP. pileus. This also finds support from experimental evidence in hydra, since TCF was found to be among the few conserved genes strongly expressed in stem cells of all three cellular lineages in this cnidarian polyp P. pileus (this study) there is at least one instance where the pathway seems activated in a differentiated context  during evolution along the ctenophore lineage. The distinctive expression patterns of the two Frizzled receptors in the basal cushion furthermore suggest that Wnt signalling regulates polarity within this structure according to two perpendicular directions, even though gradients of Wnt ligands are likely oriented only along the comb midline. Thus PpiWntA, PpiWntX and PpiWnt6 are expressed symmetrically with respect to the midline with staggered expression domains starting from the basal cushion extremity, PpiWntA having the most restricted expression zone and PpiWnt6 the broadest. One of the receptors (PpiFzB) is strongly expressed at both basal cushion extremities, symmetrically with respect to the midline, and thus probably provides target cells with information about their position along the midline, certainly a critical parameter notably for regulating cell proliferation vs. differentiation. In contrast, the other receptor PpiFzA is expressed only on the aboral side of the midline.The highly structured spatial distribution of transcripts of multiple Wnt signalling genes observed in PpiFzA-expressing cells, the sharp oral/aboral polarity of PpiFzA expression  is quite remarkable since morphologically the basal cushion is essentially symmetric with respect to the midline, notably with polster cell bodies of the oral and aboral halves being identical from a cytological viewpoint  might reflect an involvement here of the Fz-PCP pathway PpiFzA in fact integrates positional information along both spatial coordinates, since in addition to oral/aboral polarity, it also displays polarity between extremity and centre, with no expression at the latter pole, and a distinct circular spot of expression positioned at the centre .Whatever the exact nature of these iewpoint . It was e centre . This al2+ pathway, while in neural development and synapse formation the Wnts act predominantly through the Wnt/β-catenin pathway In bilaterians, Wnt signalling not only plays crucial roles during neural development PpiWnt6, PpiDvl1, PpiTcf); the eight ciliated grooves  of several key roles that this pathway endorses in “higher” animals (e.g. vertebrates) in development and tissue homeostasis: control of the main body axis through a posterior (in bilaterians)/oral (in coelenterates) signalling centre, control of morphological polarities at lower spatial scales, promotion of cell proliferation and undifferentiated state, and specific additional functions within the neuro-sensory system.P. pileus really plays a role with respect to global oral/aboral body polarity. Indeed an alternative possibility could be that Wnt/β-catenin signalling around the mouth margin would be involved only in local regulations (e.g. of cell proliferation and/or differentiation in this area). Use of pharmacological drugs to block or activate the pathway, and/or gene interference using RNAi to selectively inhibit the activity of genes involved in Wnt signalling, can also be expected to provide insights about functions of the pathway in the control of cell proliferation notably along cellular lineages of the tentacle root conveyor belts, in building specific neuro-sensory structures, and in regulating polarity and regular repetition of the basal cushions within the comb rows.The present work provides a solid platform for future experimental studies targeted at clarifying the functions of Wnt signalling in various contexts both during development and in the adult body in ctenophores. Notably, it will be important to determine whether the Wnt oral centre uncovered in the adult A further stimulating perspective will be to investigate genes associated with Wnt/Fz signalling through β-catenin independent mechanisms, such as the Fz-PCP (planar cell polarity) and Fz-Rho pathways File S1Gene phylogenetic analyses.(PDF)Click here for additional data file.File S2Negative (sense probe) and positive (PpiTbxE and PpiPax1) controls for whole mount in situ hybridisation.(PDF)Click here for additional data file.File S3DNA label incorporation experiments providing insights into cellular dynamics in the polar fields.(PDF)Click here for additional data file.File S4Gene expression in the comb rows.(PDF)Click here for additional data file.


At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated  and invasive carcinomas  and with the E-cadherin staining . Carcinoma cells with β-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-β, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-β with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of β-catenin fine-tune the carcinoma progression.The binding of p120-catenin and β-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and β-catenin in oral carcinomas ( Oral squamous cell carcinoma is a common malignant neoplasm of the head and neck and prevalence is predicted to increase in the next few decades. Regardless of therapeutic approach, location, or stage of the disease, more than 50% of patients experience a relapse . AlthougEpithelial cells develop tight cell-cell adhesions as compared to other cell-types. They form distinct tissue layers through the formation of tissue boundaries and change tissue shapes causing cell rearrangements or the conversion between histological cell states and the long-range migration of cells. The stratified squamous epithelium that covers the oral cavity is especially well organized and form strong cell-cell adhesions mediated by cadherins. Cadherins are a superfamily of calcium-dependent transmembrane proteins that are developmentally regulated and evolutionally conserved cell-cell adhesion molecules. Within the cadherin superfamily, epithelial-type E-cadherin plays a decisive role for development and maintenance of epithelium at the adherence junction .2-terminal region; isoform 1 is the longest variant and isoform 4 is the shortest variants [The cytoplasmic domain of E-cadherin binds with catenins. Catenins mediate the interaction of E-cadherin with the cytoskeleton including actin filaments and microtubules, and stabilize adherence junctions . p120-cavariants .β-catenin binds to the COOH-terminal of the E-cadherin cytoplasmic domain through the armadillo-repeats domain and mediates ligation of actin filaments . The cytBoth catenins can also support neural N-cadherin-mediated cell-cell adhesion as in E-cadherin, and E-cadherin to N-cadherin switch, enhancing carcinoma progression ,3. HowevSince p120-catenin protein consists of four isoforms, we used an antibody that can equally recognize all of them . All isoBoth catenins immunolocalized at the cell membrane region of normal oral epithelial cells from the basal to suprabasal layers and weaken the reactivity toward the cornified layer . At the P < 0.01), there was no statistical significance between the cytoplasm-positive cells at the center (18.7 ± 23.1%) and the invasive front . The percentage of membrane-positive cells inversely correlated with the cytoplasm-positive cells at the center  but not at the invasive front . The nuclear staining was not detected in this study.The p120-catenin was strongly detected at the center of the tumors but rapidly decreased toward the invasive front . Since cP < 0.01; invasive front, P = 0.01; The percentage of membrane-positive cells was decreased in parallel with tumor dedifferentiation , and the cytoplasm/nucleus-positive cells at the invasive front (47.5 ± 31.2%) were higher than that at center . An inverse correlation between membrane-positive cells and cytoplasm/nucleus-positive cells at the center  and invasive front  and TGF-β + TNF-α treatment . β-catenin expression was not affect by the treatments . Single treatment of cells with TGF-β, TNF-α or EGF did not affect their expression (data not shown).Carcinoma cells and HaCaT cells were treated with tumor stimulus, TGF-β, TNF-α or EGF. These stimuli did not apparently affect the expression of p120-catenin isoforms . HoweverCatenins stabilize adherence junctions by binding to cadherins and to cytoskeletons. E-cadherin-mediated cell-cell adhesion regulates proliferation and differentiation of epithelial cells ,19 and shigh/E-cadherinhigh and p120-cateninlow/E-cadherinlow groups, suggesting that oral carcinoma cells at the invasive front concomitantly lose p120-catenin and E-cadherin expression. Loss of p120-catenin initiates E-cadherin protein degradation [per se may define E-cadherin-mediated adhesion at the protein level independent of the transcriptional control in aggressive oral carcinomas. Although the mechanism for p120-catenin down-regulation in carcinoma tissues is not defined clearly, it is transcriptionally inactivated by ZEB2, an E-cadherin repressor that is predominantly expressed at the invasive front of oral carcinomas with worse prognosis [E-cadherin expression is inactivated by the transcriptional repressor binding and promoter hypermethylation in oral carcinomas ,22. In aradation ,28,29, bradation . Loss ofrognosis ,30. The rognosis ,32. UnveAmong p120-catenin isoforms, this study confirmed the predominant expression of isoform 3 in oral carcinoma cells and normal keratinocytes. Since previous studies suggested the involvement of isoform shift from 3 to 1 in carcinoma progression ,33, we tin vitro condition [As in a number of previous studies, β-catenin staining at the cell membrane was decreased in parallel with carcinoma dedifferentiation and diffuse invasion, but the cytoplasmic/nuclear staining was detected in almost half of carcinoma cells. β-catenin did not alter its expression under TGF-β, EGF and TNF-α treatment that decreased p120-catenin, suggesting that the mechanism for β-catenin expression is largely different from p120-catenin. β-catenin localizes at the cytoplasm and nucleus in parallel with WNT expression in oral carcinoma cells at the invasive front . In addiondition , it was In normal oral epithelium, p120-catenin expression was weak and strictly restricted at the cell membrane of basal cells. The weak expression polarizes the basal cells to secrete basement membrane proteins to the underlying tissue , and genE-cadherin expression is one of prime determinant for the carcinoma progression of epithelial origin  and it iA total of 67 individual oral carcinomas were taken at Kanazawa University Hospital from biopsies or surgeries from 1988 to 2003. The median age of the study patients was 63.7-yrs  at the time of diagnosis. The details of the pretreatment clinical and pathologic characteristics were summarized in 2 incubator. Immortalized normal keratinocyte cell line, HaCaT [Immortalized human oral carcinoma cell lines  were used . Cells we, HaCaT ,50, was Unstained formalin-fixed and paraffin-embedded sections of oral carcinomas and normal epithelium were treated with microwave (500 W) in 0.01 M sodium citrate buffer, pH 6.0, and incubated with mouse antibodies against p120-catenin  or β-catenin  followed by biotinylated secondary antibodies . After treatment with avidin-biotin complexes , the color was developed with 3,3’-diaminobenzidine tetrahydrochloride. The percentage of carcinoma cells stained at the membrane or cytoplasm were calculated at least 3,000 cells for each section randomly selected. We determined that carcinoma cells with the strong circumferential membranous staining were membrane-positive and with strong cytoplasmic granular staining were cytoplasm-positive. The percentage of carcinoma cells stained membrane and cytoplasm was independently determined according to previous studies ,51–54. TThe MB proteins and MNB proteins were isolated from oral carcinoma cells and HaCaT cells by the ProteoExtract Native Membrane Protein Extraction Kit . After size-fractionation of proteins on SDS-polyacrylamid gels under the reduction (2 µg/lane) and electrotransferring to PDVF membranes, the membranes were probed with anti-p120-catenin or -β-catenin antibodies. Anti-Na/K ATPase  and -β-actin (Sigma-Aldrich) antibodies were used for the positive controls of MB and MNB fractions, respectively. To determine the p120-catenin isoform shift by immunoblotting, cells were incubated in serum-free medium overnight, treated with human TGF-β , EGF  and TNF-α  for 48 h, and harvested the hole-cell lysates in SDS-polyacrylamide gel sample buffer. The membranes were put on a flatbed scanner, and intensity of the bands was compared using the ImageJ 1.46r .U test using JMP 7.0.1 . To analyze the percentage of catenin-positive cells and E-cadherin-positive cells at the invasive front (n = 39), the raw data for E-cadherin-positive cells were quoted from our previous study published in PLoS ONE [Association of percentage of catenin-positive carcinoma cells and the clinicopathological parameters were analyzed by Welch’s ANOVA, regression analysis or Mann-Whitney PLoS ONE  and subjTable S1Percentage of p120-catenin-membrane positive carcinoma cells and clinicopathological implications.(DOC)Click here for additional data file.Table S2Percentage of p120-catenin-cytoplasm stained carcinoma cells and clinicopathological implications.(DOC)Click here for additional data file.Table S3Percentage of β-catenin-membrane positive carcinoma cells and clinicopathological implications.(DOC)Click here for additional data file.Table S4Percentage of β-catenin-cytoplasm stained carcinoma cells and clinicopathological implications.(DOC)Click here for additional data file.Table S5Correlation of percentage of catenin and cadherin positive carcinoma cells.(DOC)Click here for additional data file.Table S6Clinicopathological parameters of 67 primary oral carcinomas.(DOC)Click here for additional data file.


The canonical WNT pathway has been implicated as playing important roles in the pathogenesis of a variety of kidney diseases. Recently, WNT pathway activity was reported to be elevated in the renal tissue of a lupus mouse model. This study aimed to evaluate the potential role of the WNT pathway in the pathogenesis of human lupus nephritis.The expression of β-catenin was evaluated in renal biopsy specimens from lupus nephritis patients and control kidney tissues by immunohistochemistry and western blotting. Real-time polymerase chain reaction (RT-PCR) was used to detect RNA expression of β-catenin, Dkk-1 and Axin2. Plasma concentrations of Dkk-1 were measured by ELISA.p<0.05), accompanied by an increase in mRNA expression of β-catenin (p<0.01) and axin2 (p<0.05).Immunohistochemistry and western blotting revealed increased expression of β-catenin in the kidneys of patients with lupus nephritis compared with control kidney tissues (p<0.01) at the mRNA expression level; the increase in β-catenin mRNA positively correlated with the creatinine clearance rate (Ccr) and negatively correlated with chronicity indices of renal tissue injury. Greater plasma Dkk-1 concentrations were found in LN patients compared with controls (p<0.05). Plasma Dkk-1 concentrations also correlated negatively with anti-dsDNA antibody levels and positively with serum C3 levels.β-catenin was significantly greater in LN patients without renal interstitial fibrosis compared with those with renal interstitial fibrosis (The canonical WNT/β-catenin signaling pathway was activated in lupus nephritis patients, accompanied by an increase in plasma levels of Dkk-1. Altered WNT/β-catenin signaling was related to the pathogenesis of lupus nephritis and might play a role in renal fibrosis. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the formation of various autoantibodies and multiorgan involvement. The kidneys are the most commonly affected organs, and renal involvement is a major cause of death in lupus patients The WNT signaling pathway is best known for its role in embryogenesis and cancer Increased WNT signaling induces the expression of matrix metalloproteinases (MMPs) This study was approved by the Review Board for Renji Hospital in Shanghai, the Republic of China. Written informed consent was obtained from all study participants. Nighty-seven consecutive patients with lupus nephritis who had been referred to Shanghai Renji Hospital  during 2010-2011 for renal biopsy, 34 control specimens, and 40 healthy donors were recruited for this study . All lupAll patients underwent an ultrasound-guided renal needle biopsy. The renal tissues obtained by biopsy were fixed in 10% neutral buffered formalin, gradually dehydrated, and embedded in paraffin. Sections were stained with hematoxylin-eosin and periodic acid-Schiff reagent using standard protocols. Biopsy specimens were classified using the International Society of Nephrology/Renal Pathology Society (ISN/RPS) 2003 classification of LN Renal tissue injury was evaluated using activity and chronicity indices as previously reported by Austin and colleagues One portion of the fresh renal tissue was snap-frozen in liquid nitrogen immediately and stored at −80°C before use. Proteins of frozen tissues were obtained with RIPA(Thermo Scientific). Proteins were then subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, blotted with the monoclonal rabbit anti-human β-catenin antibodies , and detected with Luminol/Enhancer Solution . Tubulin antibodies and conjugated secondary antibodies were obtained from cell signaling technology (USA). Relative protein levels were quantified using ImageJ software, version 2.1.4.7 One portion of the fresh renal tissue was snap-frozen in liquid nitrogen immediately and stored at −80°C before use. Total RNA in the frozen tissues was extracted using TRIzol  following the manufacturer's protocol. The quantity and quality of RNA was measured using a NanoDrop™ 1000 spectrophotometer . Sixty-four samples with 260/280 nm ratios above 1.8 were reverse transcribed into cDNAs using a Reverse Transcription System  with random primers.5′-CGCAGGCGTGCAAATCT-3′, reverse 5′-TGACGCATGCAGCGTTTT-3′; β-catenin, forward 5′-GGCCTCTGATAAAGGCTACTGTTG-3′, reverse 5′-ACGCAAAGGTGCATGATTTG-3′; Axin2, forward 5′- CGGGAGCCACACCCTTCT-3′, reverse 5′- TGGACACCTGCCAGTTTCTTT -3′, and RPL13A, forward 5′-CCTGGAGGAGAAGAGGAAAGAGA-3′, reverse 5′-TTGAGGACCTCTGTGTATTTGTCAA-3′.Quantitative PCR was performed using SYBR green dye for the detection of β-catenin and Dkk-1 mRNA expression in an ABI Prism 7900HT Sequence Detection System (Applied Biosystems). RPL13A was used as an endogenous control. The sequences of the primers were as follows: Dkk-1, forward Relative expression levels were calculated using the -ΔΔCt method. Normalization was accomplished by comparing the relative expression of each sample with that of the control sample.Plasma Dkk-1 levels from SLE patients and healthy donors were measured by enzyme-linked immunosorbent assay using a commercially available test kit  according to the manufacturer's protocol.P values under 0.05 were considered statistically significant.Statistical analysis was carried out using GraphPad Prism Software, version 5.0. The results are presented as the mean±SD. The Student's t-test and one-way analysis of variance (ANOVA) were used for determining differences between groups. For heterogeneity of variance, differences were tested with the Mann-Whitney test for the comparison of two groups. Correlations were determined by two-tailed Pearson correlation analysis. Correlation coefficient was applied as the index of measuring correlation, The distribution of the ISN/RPS classification of the 97 patients was as follows: 7 were class III, 18 were class IV, 28 were class V, 18 were class V+III, and 26 were class V+IV .Althought some result of statistical analysis was no significant,there was a trend than class IV and V+IV patients was likely to had greater SLEDAI scores, 24-hour proteinuria, and lower levels of serum complement component 3(C3) and albumin. Consistent with the clinical presentation of the disease, the class IV patients also had more disease activity.p <0.01, β-catenin was found to be expressed in the tubular compartments of the glomerulus glomeruli  A, B. Thp<0.01)  . Moreovep = 0.006).  or dosage of corticosteroid. However, gene expression of β-catenin positively correlated with Ccr and negatively correlated with CI, both of which are indicators of the degree of renal injury . In addi 0.006). . The pat 0.006). .p<0.01  plays a central role in the complement system and contributes to innate immunity. Increased circulating anti-dsDNA antibodies and decreased C3 level predicted higher lupus activity. Our data showed the plasma Dkk-1 concentration inversely correlated with the anti-dsDNA antibody concentration and positively correlated with the serum C3 concentration, indicating that the plasma Dkk-1 concentration was inversely associated with the activity of SLE.Since Dkk-1 is a downstream of gene of β-catenin, activated β-catenin signaling will lead to upregulation of Dkk-1, which might interpreted the phenomenon of positive correlation between Dkk-1 mRNA expression and expression of β-catenin in LN renal samples. Study on lupus mouse model done also showed a parallel increased levels of Dkk-1 and β-catenin in kidneys The correlation coefficients were low in our correlation analysis. The possible reason might the heterogeneity of our clinical samples and small sample size. In our future research, increasing sample size and screening patients with more homogenous backgrounds might increase the correlation coefficients.In our study, the increased levels of Dkk-1 in plasma did not correlate with Dkk-1 mRNA expression in the kidneys.β-catenin was widely expressed in the immune system, the resource of plasma Dkk-1 might be produced by multiple systems and affected by many factors expect renal.In this study, we found that the expression of β-catenin and Axin2 in the kidneys of patients with lupus nephritis was increased. These findings indicated that the classical WNT/β-catenin pathway might play a role in the pathogenesis of lupus nephritis. β-catenin mRNA expression was higher in patients without renal fibrosis than in those with renal fibrosis; additionally, gene expression of β-catenin significantly correlated with Ccr levels and CI. Dkk-1, an inhibitor of the WNT pathway, was greater in the plasma of LN patients in our study. Considering the relationship between Dkk-1, C3 and anti-dsDNA antibody, our data suggested that Dkk-1 might play a protective role in lupus nephritis. The WNT/β-catenin pathway lies upstream of factors that promote fibrosis, and thus, this pathway might be an important element in the induction of renal fibrosis and, eventually, the progression to end-stage renal disease. Further studies on the WNT/β-catenin pathway will help foster understanding of the pathogenesis of lupus nephritis and aid the search for new therapeutic targets.


Rhizobium sp. strain NGR234, which shows sequence similarities with novel E3 ubiquitin ligase (NEL) domain effectors from the human pathogens Shigella flexneri and Salomonella enterica. NopM expressed in Escherichia coli, but not the non-functional mutant protein NopM-C338A, showed E3 ubiquitin ligase activity in vitro. In vivo, NopM, but not inactive NopM-C338A, promoted nodulation of the host plant Lablab purpureus by NGR234. When NopM was expressed in yeast, it inhibited mating pheromone signaling, a mitogen-activated protein (MAP) kinase pathway. When expressed in the plant Nicotiana benthamiana, NopM inhibited one part of the plant's defense response, as shown by a reduced production of reactive oxygen species (ROS) in response to the flagellin peptide flg22, whereas it stimulated another part, namely the induction of defense genes. In summary, our data indicate the potential for NopM as a functional NEL domain E3 ubiquitin ligase. Our findings that NopM dampened the flg22-induced ROS burst in N. benthamiana but promoted defense gene induction are consistent with the concept that pattern-triggered immunity is split in two separate signaling branches, one leading to ROS production and the other to defense gene induction.Type 3 effector proteins secreted via the bacterial type 3 secretion system (T3SS) are not only virulence factors of pathogenic bacteria, but also influence symbiotic interactions between nitrogen-fixing nodule bacteria (rhizobia) and leguminous host plants. In this study, we characterized NopM (nodulation outer protein M) of  Rhizobium sp. strain NGR234. We demonstrate that NopM possesses E3 ubiquitin ligase activity, indicating that NopM can “tag" proteins with ubiquitin, and thus target them for proteasome-dependent degradation. Using a mutant approach, we demonstrate that enzymatically active NopM promotes establishment of symbiosis with Lablab purpureus, the host plant from which NGR234 was originally isolated. We further examine effects of NopM when directly expressed in eukaryotic cells and show that NopM interferes with specific signaling pathways. NopM expressed in the model plant Nicotiana benthamiana dampened generation of reactive oxygen species (ROS), which are formed in response to the bacterial flagellin peptide flg22. We suggest that NopM promotes nodule initiation by reducing the levels of harmful ROS during the infection process.Many Gram-negative bacterial pathogens possess type 3 secretion systems, which deliver effector proteins into eukaryotic host cells through needle-like structures. Effectors manipulate the host cell and many of them suppress host defense responses. Interestingly, certain symbiotic strains of rhizobia also possess such secretion systems. Rhizobia infect legume roots and induce root nodules, where the bacteria convert atmospheric nitrogen into ammonia. Here, we characterize the effector NopM of  Type 3 effector proteins of pathogenic Gram-negative bacteria are transported into eukaryotic host cells through the bacterial type 3 secretion system (T3SS), which forms a needle-like pilus Parasponia. As a result of rhizobial infection, roots of host plants develop nodules, in which the bacteria differentiate into bacteroids. For the host's benefit, atmospheric nitrogen is then reduced to ammonia by the bacterial nitrogenase enzyme. During nodule formation, various signal molecules are exchanged between the two partners Rhizobium sp. strain NGR234 ttsI. This gene encodes a transcriptional activator, which controls expression of genes that have a conserved cis-element in their promoters, named tts-box. In NGR234 and a number of other strains, genes encoding a bacterial type 3 secretion system (T3SS) and corresponding type 3 effectors are regulated by TtsI Interestingly, certain rhizobia also use type 3 effectors during symbiosis with host legumes Lotus japonicus, NopL suppressed expression of defense genes nopL rapidly developed necrotic areas, indicating a lack of suppression of defense Rj2 and Rfg1) of certain soybean cultivars are involved in host-specific nodulation and prevented establishment of symbiosis with specific strains in a T3SS-dependent manner One approach to study the function of bacterial effectors is to express them singly in eukaryotic cells. The type 3 effector proteins NopL and NopT (nodulation outer proteins L and T) of strain NGR234 have been characterized in this way. When expressed in tobacco and Sinorhizobium fredii HH103 using a proteomic approach, in which secreted proteins from a T3SS-deficient mutant were compared to proteins from wild-type bacteria Rhizobium sp. strain NGR234 (nopM formerly y4fR), Bradyrhizobium japonicum USDA110 (blr1904 and blr1676) and B. elkanii USDA 61 (nopM). During the course of the present study, T3SS-dependent secretion of NopM has been reported for strain NGR234 and a nopM deletion mutant induced fewer nodules on the host Lablab purpureus compared to the parent strain nopM promoter activity depended on TtsI, which is predicted to bind to the conserved tts box (TB1) in the promoter region of nopMShigella flexneri (such as IpaH9.8 and IpaH1.4) and Salomonella enterica  Yersinia pestisPseudomonas syringae and Ralstonia solanacearum (e.g. HpX29 of R. solanacearum strain RS1000) The leucine-rich repeat (LRR) protein NopM (nodulation outer protein M) was first identified in S. flexneri (such as IpaH9.8 and IpaH1.4) and S. enterica  IpaH family effectors are E3 ubiquitin ligases with a NEL (novel E3 ligase) domain. Enzymatic activity has been demonstrated for effectors from of S. enterica blocks the innate immune system of human cells by interfering with the nuclear factor κB (NF-κB) pathway. IpaH9.8 interacts with NEMO  complex) and the ubiquitin-binding adaptor protein ABIN-1. As a result, NEMO is polyubiquitinated and NF-κB activation is suppressed S. enterica targets thioredoxin and ERdj3, an endoplasmic reticulum luminal chaperone Bacterial E3 ubiquitin ligases delivered into host cells mimic the activities of host E3 ubiquitin ligases and ubiquitinate specific target proteins. For example, IpaH9.8 Rhizobium sp. strain NGR234. We demonstrate that NopM possesses E3 ubiquitin ligase activity. Our mutant analysis reveals that NopM acts as an E3 ubiquitin ligase during symbiosis with the host L. purpureus. NopM activity also inhibits mating pheromone signaling when expressed in yeast, and MAMP-triggered generation of reactive oxygen species (ROS) when expressed in Nicotiana benthamiana plants, while stimulating expression of MAMP-induced defense genes at the same time. We discuss our results in the light of the role of NopM in symbiosis.In this study, we characterize NopM of nopM was cloned into pET28b resulting in plasmid pET-nopM. A second plasmid, pET-nopM(C338A), was constructed in which the cysteine residue 338 of NopM was replaced by alanine. Residue C338 in the C-terminal NEL domain is predicted to be an essential catalytic residue required for the ubiquitin transfer Escherichia coli BL21 (DE3) harboring the constructed plasmids were induced with IPTG and extracted proteins were purified using nickel–nitrilotriacetic acid affinity chromatography. When analyzed by SDS-PAGE, a strong band with an apparent molecular mass of about 65 kD was detected, which corresponded to His-tagged NopM and NopM-C338A, respectively . This band was not seen, when proteins from E. coli BL21 (DE3) harboring the empty vector pET28b were purified in a similar way. Immunoblot analysis revealed that corresponding anti-NopM antibodies recognized His-tagged NopM and NopM-C338A proteins , was constructed by using a corresponding DNA sequence encoding NopM-C338A (nopM(C338A) mutant, respectively. As expected, no bands were seen for the knock-out mutant NGRΩnopM. Strain NGRΩrhcN, a mutant lacking a functional T3SS served as a negative control nopM carrying the plasmid pFAJ-nopM (containing nopM including its promoter sequence) secreted NopM, indicating complementation by this plasmid (Two mutant derivatives of pM-C338A . Protein plasmid .L. purpureus was inoculated. nopM induced significantly fewer nodules (1–2 nodules per plant), indicating that NopM was required for optimal nodulation of this host plant. Plants inoculated with NGRnopM(C338A) showed a similar reduction in nodulation, indicating that the C338 residue is essential for the nodule-promoting effect of NopM. The symbiotic phenotype of NGRΩnopM and NGRnopM(C338A) on L. purpureus could be complemented when plasmid pFAJ-nopM was introduced into these mutants. The nodule number was significantly increased and reached values comparable to those of the parent strain NGR234 (Nodulation phenotypes of the examined strains differed when the legume n NGR234 .Phaseolus vulgaris (cv. Yudou No 1). Optimal nodulation of this plant with NGR234 required NopT, another type 3 effector of NGR234 nopM or NGRnopM(C338A) were similar to those obtained from the parent strain NGR234, however. Similarly, nodulation tests with the constructed mutants showed no obvious differences for Flemingia congesta (data not shown), although nodulation of this host plant is improved by a functional T3SS Nodulation tests were also performed with nopM mutants depended on the tested host legume and that the positive effect of NopM on L. purpureus nodulation likely depended on its E3 ubiquitin ligase activity.Taken together, the mutant analysis revealed that the symbiotic phenotype of the constructed S. flexneri blocked mating pheromone (α-factor) response signaling, a specific MAP kinase pathway MATa) results in a typical halo of growth inhibition nopM and the point-mutated sequence encoding NopM-C338A were cloned into the expression vector pESC-leu, which has a galactose-inducible promoter (GAL1). W303-1A cells carrying the resulting plasmids (pESC-nopM and pESC-nopM(C338A), respectively) expressed NopM and NopM-C338A on galactose plates: An immunoblot with anti-NopM antibodies exhibited a band corresponding to the predicted size of NopM (60.5 kD), which was absent in cells transformed with the empty vector pESC-leu  or the empty vector pESC-leu. Yeast transformed with pESC-nopM showed normal growth on SD/galactose plates. In contrast, cells transformed with pESC-nopM(C338A) or pESC-leu poorly grew on SD/galactose . In disks expressing NopM-C338A, however, the ROS burst in response to flg22 was similar to empty vector controls (p = 0.24). Accordingly, differences between disks expressing NopM and NopM-C338A were significant , suggesting that ROS suppression depends on the ubiquitin E3 ligase activity of NopM (Transient generation of reactive oxygen species (ROS) induced by MAMPs is a rapid signaling response, which depends on Rboh enzymes (respiratory burst oxidase homologs) and is activated by calcium-dependent protein kinases (CDPKs)  of NopM .NbAcre31  and NbCyp71d20 (encoding a putative cytochrome P450) are upregulated in response to flg22 NbAcre31 , but not of NbCyp71d20 (p = 0.08). In contrast, cells expressing NopM-C338A neither showed increased transcript levels of NbAcre31 nor of NbCyp71d20. As expected, leaf tissue challenged with flg22 showed stimulated expression, particularly for NbCyp71d20. When compared to empty vector controls, effects of the flg22 treatment on NbCyp71d20 activation were significantly stronger in either NopM or NopM-C338A expressing tissues  N. benthamiana were visualized on immunoblots with anti-p42/44-phospho-ERK antibodies, which recognize activated SIPK and WIPK (wound-induced protein kinase). Expression of NopM in N. benthamiana did not result in MAP kinase activation. A treatment of leaves with flg22 for 15 min caused MAP kinase activation in control plants transformed with the empty vector as well as in NopM or NopM-C338A expressing plants, indicating that NopM did not block flg22-induced MAP kinase signaling  domain effectors. These enzymes are structurally unrelated to other bacterial E3 ubiquitin ligases, which have a HECT or RING/U-box domain nopM and NGRnopM(C338A), showed reduced nodulation on L. purpureus, indicating the importance of the C338 residue during establishment of symbiosis. We suggest that NopM functions as an E3 ubiquitin ligase during the infection process and that ubiquitination of one or more host proteins helps to promote nodulation on L. purpureus. In some other host plants, however, effects of NopM on nodulation were either not observed or even negative  mediated ROS production Yeast is a model to investigate effects of type 3 effector proteins in eukaryotic cells N. benthamiana depended on the C338 residue of NopM, suggesting ubiquitination of a MAMP signaling component. The E3 ubiquitin ligase activity of NopM is reminiscent of the virulence function of AvrPtoB, a type 3 effector of P. syringae with a functional E3 ubiquitin ligase RING/U-box E3 domain Arabidopsis thaliana, AvrPtoB targets proteins such as the flagellin receptor complex FLS2-BAK1 N. benthamiana promoted flg22-triggered accumulation of NbCyp71d20 transcripts. Hence, ubiquitination activity of NopM was not essential to induce this effect. We suggest that an interaction between NopM and a N. benthamiana protein is sufficient to cause a partial deregulation of immune signaling in flg22-challenged tissue.Suppression of flg22-triggered ROS in nopM(C338A) suggest that the E3 ubiquitin ligase activity of NopM is required for optimal nodulation of the host plant L. purpureus. When expressed in N. benthamiana, NopM suppresses the flg22-elicited ROS burst, suggesting that NopM blocks ROS-associated defense responses. Future work is required to test whether NopM can also suppress ROS formation in legume roots. Indeed, ROS generation could be detrimental during the rhizobial infection process L. pupurpureus infection threads below a harmful threshold level.Taken together, we provide genetic and biochemical evidence that NopM is a type 3 effector with a functional NEL domain. Inoculation tests with the constructed point mutant NGRBacterial strains and plasmids used for this study are listed in Table S1 of Rhizobium (Sinorhizobium fredii) sp. NGR234 (accession number AAB91674) was cloned into the pET28b vector, resulting in plasmid pET-nopM. PCR-based site-directed mutagenesis was used to mutate the cysteine 338 (TGT codon) of NopM into alanine (GCT codon) and the resulting plasmid was named pET-nopM(C338A). The plasmids were then transformed into E. coli BL21 (DE3) cells. The His-tagged NopM and NopM-C338A proteins were purified from isopropyl-β-D-thiogalactopyranoside (IPTG) induced cultures by nickel affinity chromatography with Ni-NTA resin beads . For immunization of a New Zealand rabbit, Ni-NTA purified His-tagged NopM was separated by SDS-PAGE and gel bands containing NopM were cut from the gel.The sequence encoding NopM of E. coli cultures grown at 27°C for 12 h were purified by nickel affinity chromatography according to the manufacturer's recommendations under non-denaturing conditions . Ubiquitination assays were performed in a 40-µl volume containing the reaction buffer (25 mM Tris HCl (pH 7.5), 50 mM NaCl, 5 mM ATP, 10 mM MgCl2, 0.1 mM DTT), 2 µg HA-ubiquitin, 0.5 µg of E1, and 2 µg of UbcH5B in the presence or absence of 1 µg of His-tagged NopM, or NopM-C338A, respectively. Reactions were incubated at 37°C for 1 h and stopped by addition of an equal volume of Laemmli sample buffer (62.5 mM Tris HCl (pH 6.8), 10% (v/v) glycerol, 2% (w/v) SDS, 0.005% (w/v) bromophenol blue) containing 100 mM DTT. Reaction mixtures were separated by SDS-PAGE, transferred onto a nitrocellulose membrane, and probed with specific antibodies  at 1∶ 4 000 dilution). Immunoblots were developed with enhanced chemiluminescence reagents .Purified ubiquitin-activating enzyme from human (E1), UbcH5B (E2), and HA-ubiquitin were purchased from Boston Biochem . His-tagged NopM and NopM-C338A from nopM, a 2.5-kp fragment containing nopM was cloned into pBluescript II KS(+), generating pSK-nopM2500. PCR-based site-directed mutagenesis was used to generate a BamHI restriction site close to the ATG codon of nopM. A spectinomycin-resistant (Spr) Ω interposon was excised from pHP45 BamHI and ligated into the BamHI site, generating pSK-nopMΩ. The construct was then cloned into the suicide vector pJQ200SK nopMΩ) was mobilized from E. coli DH5α into Rhizobium sp. NGR234 by triparental mating using the pRK2013 helper plasmid r) and for growth on 5% (w/v) sucrose. The obtained mutant NGRΩnopM was confirmed by Southern blot analysis using the DIG DNA labeling and detection kit as specified by the supplier .For construction of the mutant NGRΩnopM(C338A), plasmid pSK-nopM2500 was mutated by a PCR-based site-directed mutagenesis approach, thereby creating the restriction site Aor51HI. The insert of this plasmid (named pSK-nopM(C338A)) with DNA encoding NopM-C338A was then cloned into the suicide vector pJQ200SK, resulting in plasmid pJQ-nopM(C337A). After conjugation, Rhizobium sp. NGR234 bacteria were first cultivated on agar plates containing gentamycin and rifampin and then on plates containing rifampin and 5% (w/v) sucrose. Genomic DNA from candidate colonies served as a template for a PCR  was completely cleaved by Aor51HI into two smaller fragments.For construction of NGRnopM was cloned into pFAJ1702 nopM) was then mobilized into Rhizobium sp. NGR234 and selection was performed on agar plates containing tetracycline.For complementation analysis of the constructed mutants, a 2081-bp fragment containing the coding region and promoter sequence of Rhizobium sp. strains NGR234 (parent strain), NGRΩnopM (this study), NGRnopM(C338A) (this study), NGRΩnopM carrying pFAJ-nopM (this study) and NGRΩrhcNSecreted proteins from culture supernatants from Lablab purpureus cv. Chaojibiandou, Phaseolus vulgaris cv. Yudou No 1, and Flemingia congesta. Seeds were surface sterilized and germinated on agar plates, and plantlets were transferred to 300-mL plastic jar units linked with a cotton wick . Plants (1 plant per jar) were inoculated with 109 bacteria .Nodulation tests were performed in plastic jars using the host plants Saccharomyces cerevisiaeMATa) strain SY2227) and constructed plasmids encoding NopM or NopM-C338A are listed in Table S1 of 2O) to the center of each agar plate. The plates were sealed, incubated at 27°C for 1 week and then photographed.Standard media and techniques were used for transformation, maintenance, and growth of nopM, pCAMBIA-nopM(C338A), the empty vector pCAMBIA-T and pGWB417-HopQ1-myc; see Table S1 in Agrobacterium tumefaciens strain GV3101 by heat shock. Leaves from 4-week old Nicotiana benthamiana plants were infiltrated with bacteria (OD600 = 0.5) re-suspended in infiltration buffer . Expression of NopM was detected by immune blot analysis with anti-NopM antibodies at a 1∶1000 dilution. Blots were developed with CDPstar reagents (New England Biolabs). Staining of N. benthamiana leaves was performed with trypan blue as described previously Plasmids (pCAMBIA-2) were floated on water overnight and ROS released by the leaf tissue were measured using a chemiluminescent assay P≤0.05 as significantly different.Leaf discs  were then frozen in liquid nitrogen and proteins were extracted in 100 µl extraction buffer  for 30 min at 4°C. Subsequently 100 µl Lämmli loading buffer (2×) was added to each sample. Samples were subjected to immunoblot analysis using the anti-p42/44-phospho-ERK antibody (Sigma-Aldrich). Blots were developed using CDP-star technology (NEB).Two days post N. benthamiana leaf discs expressing NopM, NopM-C338A or the empty vector (EV) control were collected, and then floated overnight in water. Leaf discs were subsequently treated with 1 µM flg22 or mock-treated with BSA/NaCl solution for 30 min and then frozen in liquid nitrogen. Total RNA was extracted using the NucleoSpin RNA Plant extraction kit (Machery-Nagel). The absence of genomic DNA was checked by PCR amplification of the housekeeping NbEF1α gene by using 1 µg of RNA (NbEF1α amplification crosses an exon/intron boundary). For analysis of gene expression, first-strand cDNA was synthesized from 1 µg of RNA using AMV reverse transcriptase (Promega) and an oligo (dT) primer (Microsynth), according to the manufacturer's instructions. For quantitative PCR, 5 µl of a 1/100 µl dilution of cDNA were combined with SYBR master mix. PCRs were performed in triplicates with the 7500 Real Time PCR system (Applied Biosystems). Data were collected and analyzed with the respective ABI analyzing program. The NbEF1α RNA was analyzed as an internal control and used to normalize the values for transcript abundance. All samples were related to the empty vector (EV) control. Primers for the genes NbCyp71D20, NbAcre31 and NbEF1α are listed in Table S2 of P≤0.05 as significantly different.One day post infiltration, Rhizobium (Sinorhizobium fredii) strain NGR234: AAB91674NopM of Text S1N. benthamiana expressing NopM and NopM-C338A by immunoblot analysis, trypan blue based cell death staining and MAP kinase activation in response to flg22). (PDF)Click here for additional data file.