The inability of targeted BRAF inhibitors to produce long-lasting improvement in the clinical outcome of melanoma highlights a need to identify additional approaches to inhibit melanoma growth. Recent studies have shown that activation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma. Therefore, the identification of Wnt/β-catenin regulators may advance the development of new approaches to treat this disease. In order to move towards this goal we performed a large scale small-interfering RNA (siRNA) screen for regulators of β-catenin activated reporter activity in human HT1080 fibrosarcoma cells. Integrating large scale siRNA screen data with phosphoproteomic data and bioinformatics enrichment identified a protein, FAM129B, as a potential regulator of Wnt/β-catenin signaling.  Functionally, we demonstrated that siRNA-mediated knockdown of FAM129B in A375 and A2058 melanoma cell lines inhibits WNT3A-mediated activation of a β-catenin-responsive luciferase reporter and inhibits expression of the endogenous Wnt/β-catenin target gene, AXIN2. We also demonstrate that FAM129B knockdown inhibits apoptosis in melanoma cells treated with WNT3A. These experiments support a role for FAM129B in linking Wnt/β-catenin signaling to apoptosis in melanoma. Malignant melanoma has a poor prognosis with a 5-year survival rate of only 15%3. The recently approved therapeutic, vemurafenib, extends median patient survival by 7 months6. This major advance raises expectations that even greater rates of survival might be attainable with combination therapies.The incidence of melanoma continues to rise across the U.S. at a rate faster than any other cancer12. Analysis of melanoma tumor samples show a positive correlation between nuclear β-catenin staining and decreased tumor depth, increased patient survival and increased time to metastasis9. Moreover, treatment with WNT3A-containing conditioned media or stable overexpression of WNT3A in mouse B16 or human A375 melanoma cells reduces cell numberin vitro11. Allografts of mouse B16 or mouse xenografts of human A375 cells overexpressing WNT3A decrease tumor size compared to control11. Recently, we found that activation of Wnt/β-catenin signaling concurrent with the inhibition of the ERK/MAPK pathway synergistically elevates apoptosis in a subset ofBRAF- andNRAS-mutant cultured human melanoma cells12. Given the interaction between Wnt/β-catenin signaling and pathways known to be critical for melanoma pathogenesis, the identification of Wnt/β-catenin regulators might prove to be informative in developing novel approaches to treat this disease.Activation of the Wnt/β-catenin pathway decreases tumor growth and cooperates with ERK/MAPK pathway inhibitors to promote apoptosis in melanoma13. FAM129B is known to inhibit TNFα-dependent apoptosis in HeLa cells14. FAM129B is expressed in melanoma and promotes tumor cell invasion into collagen matrices in an ERK/MAPK phosphorylation-dependent manner13. In the present study we demonstrate that FAM129B promotes Wnt/β-catenin signal transduction in melanoma cells and that reducing levels of FAM129B with siRNA reduces the ability of WNT3A to increase apoptosis in melanoma cells.In the present study, we identify novel regulators of Wnt/β-catenin signaling in melanoma by performing a large-scale small-interfering RNA (siRNA) screen of a Wnt/β-catenin responsive reporter in human HT1080 fibrosarcoma cells, and by identifying siRNA targets that are also regulated by ERK/MAPK signaling and that have been previously associated with melanoma. By integrating these three approaches, we identified FAM129B as a potential regulator of Wnt/β-catenin signaling. FAM129B is a 746 amino acid protein that contains an amino-terminal pleckstrin homology (PH) domain and a differentially phosphorylated carboxy-terminal region15. We screened 28,044 pools of siRNAs. 19,490 gene products were targeted by one or more siRNA pool. Cells were transfected with siRNAs and treated with WNT3A-conditioned media to activate the reporter. BAR activity was normalized to the activity ofRenilla luciferase driven by the constitutive TK promoter to control for total cell number. siRNAs targeting positive control proteins such as the known Wnt/β-catenin inhibitor,AXIN2, modulated BAR activity by at least 2.0 fold with a p-value less than 0.01 an 0.01 . Using tan 0.01 .FAM129B) encoded a protein that was differentially phosphorylated following MEK inhibition .Click here for additional data file.FAM129B. First, we confirmed that all three siRNAs inhibit expression of FAM129B protein in HT1080 fibrosarcoma, A2058 melanoma and A375 melanoma cells . As previously reported12, A375 cells treated with control siRNA and the combination of WNT3A and PLX4720 exhibit robust levels of cleaved caspase-3  in these three cohorts.Given thatA and B . These d17. Our preliminary research validating these FAM129B protein-protein interactions has not yet identified the interaction responsible for regulation of Wnt/β-catenin signaling. These protein-protein interaction data are provided for future research into FAM129B cellular function.FAM129B has no known enzymatic domains and therefore likely exerts its cellular effects via protein-protein interactions. In the interest of identifying the mechanism of regulation of Wnt/β-catenin signaling by FAM129B, we identified FAM129B interacting proteins by affinity purifying an N-terminal Flag- and GFP-tagged FAM129B protein and its interactors from A375 melanoma cells. We subsequently identified interacting proteins by mass spectrometry . We identified 18 FAM129B interacting proteins, including the previously identified FAM129B interacting protein KEAP1 17. Our Data for Figure 2b. Measured β-catenin dependent firefly: constitutive renilla ratios for HT1080, A375, and A2058 reporter cells treated with the indicated conditions. Data for Figure 2c: Quantitative RT-PCR of β-catenin dependent Axin2 mRNA and housekeeping actin mRNA for HT1080, A375, and A2058 cells treated with the indicated conditions. Data for Figure 3a. Measured β-catenin dependent firefly: constitutive renilla ratios for A375 reporter cells treated with a dose curve of WNT3A or a single dose of WNT3A. Data for Figure 3b. Measured NFκB dependent firefly: constitutive renilla ratios for A375 reporter cells treated with a dose curve of TNFalpha or a single dose of TNFα . Data for Figure 4b. Quantitation of DAPI and TUNEL positive nuclei from A375 cells treated with WNT3A, PLX4720, and the control, FAM129B, or CTNNB1 siRNA.Click here for additional data file.FAM129B is required for Wnt3A to activate a β-catenin dependent reporter and reduces the ability of Wnt3A to enhance the expression of the β-catenin target geneAXIN2. We demonstrated that loss of function of FAM129B inhibits the apoptosis of melanoma cells induced by the combined treatment with WNT3A and PLX4720.We combined phosphoprotoemics and siRNA screening to identify novel regulators of Wnt/β-catenin signaling in human melanoma. We focused on FAM129B, a previously identified protein that has not formerly been linked to Wnt/β-catenin signaling. Using independent siRNAs, we confirmed that9, slows the growth of melanoma xenograftsin vivo, and cooperates with inhibition of the ERK/MAPK pathway to promote apoptosis of melanoma cellsin vitro12. There is accumulating data suggesting that Wnt/β-catenin signaling is a key regulator of melanoma metastasis. Wnt/β-catenin signaling has been implicated as the key phenotypic switch that can regulate whether melanoma cells exhibit either a proliferative or an invasive phenotype18. In mouse models of melanoma, Wnt/β-catenin signaling was recently identified as a key regulator of the metastatic phenotype18. Interestingly, FAM129B was originally identified as a protein that promotes the invasion of melanoma cells following regulated phosphorylation by the MAPK pathway13, and our own analysis showing an increased expression of FAM129B in more “invasive” melanoma cohorts supports the important role of FAM129B in melanoma suggested by this initial observation  or Nuclear Factor Kappa B (5´-GGGAATTTCC-3´) signaling pathways separated by distinct 5-base pair linkers upstream of a minimal promoter and the firefly luciferase open reading frame. The reporters also contain a separate PGK (phosphoglycerate kinase) promoter that constitutively drives the expression of a puromycin resistance gene for mammalian cell selection. These reporters were generated by Travis L. Biechele in the lab of Randall T Moon as previously published19.Detailed information on the β-catenin activated reporter plasmid (pBARLS) has been previously described15. Cell lines were cultured in a Thermo Forma steri-cult humidified incubator  at 37°C and 5% CO2. All cell lines were cultured in Dulbecco’s Modified Eagle’s Medium  containing 10% fetal bovine serum and 1% Penicillin/Streptomycin , except A375 cells, which were grown in DMEM containing 5% FBS and 1% P/S.Human A375 and A2058 cells were a generous gift from Cassian Yee . HT1080 cell lines were purchased from the American Type Culture Collection . Stable reporter lines were generated as previously described20. To monitor reporter activity and transcript activity, cells were treated with 10% WNT3A CM or LCM overnight before proceeding to subsequent assays. To monitor effects on apoptosis, cells were treated with 1% LCM or WNT3A and DMSO  or 2 uM PLX4720 .Control (LCM) and WNT3A-conditioned media (WNT3A CM) used to activate the Wnt/β-catenin signaling pathway were prepared as previously described21, with minor modifications. Briefly, HT1080 cells stably transduced with BAR firefly luciferase andRenilla luciferase lentivirus were reverse-transfected in 1536-well plates, with a final concentration of pooled siRNA at 25 nM. 48 hours after reverse transfection, cells were treated with WNT3A-conditioned media. Following overnight incubation, β-catenin dependent transcription was measured by assaying firefly luciferase activity and normalized by monitoring constitutively expressedRenilla luciferase activity as described in the Promega Dual glo luciferase assay system technical manual . All siRNAs were designed with a proprietary algorithm22.The large-scale siRNA screen was performed as previously describedFAM129B were designed using the BLOCK-iT RNAi designer and are described below. The sequence for "FAM129B A" is UCACGGACAUGAACCUGAACGUCAU. The sequence for "FAM129B B" is ACUGAGGUGCGAGAUGUCUUCUUCA. The sequence for "FAM129B C" is CAGCAGCGAUUUGAUGUGUCCAGCA. As a positive control for inhibition of Wnt/β-catenin signal transduction by siRNA, we used silencer select siRNA targetingCTNNB1 with the sequence GGUGGUGGUUAAUAAGGCUTT .Approximately 200,000 A375, A2058, or HT1080 cells (as estimated by hemocytometer counts) were reverse transfected at a final dose of 20 nM siRNA in 6-well format using 5 µl RNAi max/well . Medium GC universal stealth control siRNA was used as a negative control . Invitrogen’s stealth siRNA targeting24 hr after siRNA transfection, cells were plated in 96-well plates at a density of 20,000 cells/well. Twenty-four hours after plating, cells were treated with the indicated conditions, and luciferase activity was measured 15 hours later with a Dual-Luciferase Reporter Assay kit  and an Envision multilabel plate reader  according to the manufacturer’s suggestions.AXIN2 F" CTCCCCACCTTGAATGAAGA and "AXIN2 R" TGGCTGGTGCAAAGACATAG; and, "ACTB F" AGAGCAAGAGAGGCATCCTC and "ACTB R" CTCAAACATGATCTGGGTCA.24 hr after siRNA transfection, cells were split into a 12-well cluster plate at approximately 50% confluency. 24 hr later, cells were treated with WNT3A- or L-conditioned media. After overnight treatment, RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen). 1 µg of RNA was reverse transcribed using Fermentas’ RevertAid M-MuLV Reverse Transcriptase . QPCR was performed on a Lightcycler 480  using Lightcycler 480 DNA SYBR Green 1 master mix . The following primers were used for qPCR: "g, rinsed once with PBS, and lysed with the RIPA buffer collected from the plate lysis. Cell lysates were cleared by centrifugation at 20,000g at 4°C for 10 minutes. Protein lysates were separated by SDS-PAGE using NuPAGE 4%–12% Bis-Tris gels  in MES buffer, and transferred onto a nitrocellulose membrane  using IDEA scientific GENIE transfer apparatuses . Blots were probed using polyclonal rabbit anti FAM129B , monoclonal mouse anti Tubulin , monoclonal mouse anti β-catenin , polyclonal Rabbit anti cleaved-caspase-3 , Rabbit anti ERK1/2 , Rabbit anti phospho ERK1/2 .To test for siRNA knockdown, replicate cell lysates from low throughput reporter assays were pooled and treated with 10× RIPA lysis buffer . For monitoring cleaved caspase-3, 90% confluent 12-well plates were treated for 24-hr with the indicated conditions described in the "cell lines and cell culture" section. Media were collected and cells were rinsed once (gently) with PBS. Cells were lysed on-plate in 100 µl 1× RIPA buffer containing protease and phosphatase inhibitors . Cells were disrupted by scraping of a 1000 µl pipette tip against the plate. Apoptotic cells present in the media and PBS wash were centrifuged at 300l-lysine in a 24-well dish, rinsed with PBS, and dried. Following reverse transfection as described above, cells were seeded at a density to achieve 90 to 100% confluency at harvest. Twenty-four hours after seeding, cells were treated with the indicated conditions and incubated for 24 hours with the indicated conditions as described above in the "cell lines and cell culture" section. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed using anin situ cell death detection kit . Briefly, the medium was gently aspirated, to keep apoptotic bodies on the slide, and cells were fixed in 4% paraformaldehyde for 1 hour at room temperature. Cells were gently rinsed twice with PBS and permeabilized with 0.1% Triton X-100  in 0.1% sodium citrate  for 2 min on ice. Cells were rinsed twice with PBS and 40 ml of TUNEL reaction mixture was added directly on top of the slide; cells were incubated for 1 hour at 37°C in a humidified incubator. Slips were rinsed three times with PBS and mounted on Superfrost Plus glass slides with ProLong Gold anti-fade mounting medium containing 4´,6-diamidino-2-phenylindole (DAPI) . Images were obtained on a Nikon TiE inverted wide-field high-resolution microscope. DAPI and TUNEL positive nuclei were quantified blinded for 5 fields per slide using NIS elements .Glass coverslips were coated with poly-Except where indicated, a student’s t test was used to assess the statistical significance of the differences between the different groups; a p value of <0.05 was considered significant.24. Briefly, cleared protein extracts generated from 1 × 109 A375 cells stably transduced Flag-GFP-FAM129B were incubated with 20 μl Anti-Flag M2 affinity gel  at 4°C for 4 hr. After extensive washing, interacting affinity gel was treated with sequencing grade trypsin  and analyzed by tandem mass spectrometry as described previously23. Interacting proteins also copurified by a Flag-GFP control or commonly copurified by unrelated Flag-affinity purifications were subtracted from the final protein-protein interaction list.Flag-GFP-FAM129B and associated proteins were isolated from A375 cells as described previously I am satisfied with the changes made by the author(s).I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. I confirm that the manuscript is now approved.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. 1. The title is appropriate for the study.2. The experimental design and methods are appropriate for the topic being studied.3. The information and data provided is enough for replicating the experimentin vitro.4. The conclusions are correct but need additional data to substantiate it. In this article, the authors have identified FAM129B as a positive regulator of Wnt/beta-catenin signalling in melanoma cells. They also demonstrate that FAM129B is required for Wnt3a-induced apoptosisin vivo in animal models?Can this effect be replicatedDoes FAM129B expression correlate with beta-catenin activation and disease progression in melanoma patients?What is the mechanism by which FAM129B regulates Wnt signalling? I would like the authors to do a bit more work on answering at least one of the three questions raised. They should also discuss the other two questions in the discussion section. From my standpoint it seems the easiest thing to do would be to compare FAM129 expression in various tumor samples and correlate them to Beta-catenin activation  and growth pattern (tumor depth).However; there are three important pieces of information missing that the authors should address:I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Dear Dr. Apte,“Does FAM129B expression correlate with beta-catenin activation and disease progression in melanoma patients?” FAM129B and expression of the established Wnt/β-catenin target gene,AXIN2, within this cohort (data not shown). We have included a brief discussion of these results in our revision in order to clarify the fact that we do not have data that supports a clear role of direct regulation of Wnt/β-catenin signaling by FAM129B in melanoma patients.This is a very interesting question, and one which we had sought to address using previously published data from defined cohorts of melanoma patients. We analyzed the abundance of FAM129B present in three different cohorts of melanoma  and observed a significantly higher abundance of FAM129B mRNA in a melanoma cohort associated with higher invasiveness and relatively lower Wnt/β-catenin signaling. From this observation, we concluded that FAM129B abundance did not predict Wnt/β-catenin signaling in melanoma patients. This conclusion was supported by a lack of significant correlation between levels of “Can this effect be replicated in vivo in animal models?” We did attempt to address this question, but despite repeated attempts we were unable to stably knock down FAM129B using lentiviral shRNA in melanoma cells.  As a result, we were not able to generate cell lines that could be used to address this question in murine xenografts. “What is the mechanism by which FAM129B regulates Wnt/β-catenin signaling?” Unfortunately, we have been unable to solve the specific mechanism by which FAM129B regulates Wnt/β-catenin signaling.  We had hoped that a proteomics-based approach identifying proteins that interact with FAM129B might prove informative, but to date we have not solidified any mechanistic links based on the hits identified with this strategy.  While our efforts to validate this network are currently too preliminary for publication, we have (in the spirit of open access) included our FAM129B protein interaction network in its entirety in order to allow other readers to potentially gain insight and generate hypotheses regarding the role of this protein in regulating cellular signaling . We appreciate your thorough reading of our manuscript, as well as the thoughtful questions that you have raised in your review.  We have outlined a response to specific questions below: This report describes the identification of FAM129B as a novel regulator of Wnt/β-catenin signalling and apoptosis in melanoma cells. The FAM129B gene was identified via integration of 3 datasets, including Wnt signaling modifiers, melanoma-associated genes and the MEK/ERK phosphoproteome. FAM129B was the only gene that overlapped with all three datasets, making it a promising candidate for regulation of both Wnt and MEK/ERK pathways in melanoma. I find this an interesting report which will provide a good starting point for further studies. Overall, the data are convincing and support the conclusions drawn. Experiments which validate FAM129B as a Wnt pathway modifier  appear solid and display robust effects. The effects of FAM129B on apoptosis , however, are somewhat lower than expected based on the effects of FAM129B knockdown in reporter assays and on the inhibition of caspase cleavage. I feel that the authors could provide a more in-depth discussion on their findings and place them in the context of the background information provided in the introduction part; How do the authors consider this candidate relevant for melanoma treatment? How do the authors integrate the beneficial effects of enhanced Wnt responsiveness (this report) and the previously described enhanced tumour cell invasion (ref 20) of FAM129B expressing melanoma cells?It would be helpful for the reader to include a model which shows how Wnt and ERK pathways modulate apoptosis in melanoma cells and where FAM129B would be placed in these events. Also in  the legend of Fig. 2a it remains unclear whether endogenous levels of FAM129B are depicted.I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Dear Dr. Maurice,“How do the authors consider this candidate relevant for melanoma treatment?”It is unclear how FAM129B is relevant for melanoma treatment at this point.  Our data identifies FAM129B as a regulator of Wnt/β-catenin dependent phenotypes in melanoma cells, while previous studies had already identified FAM129B as a downstream protein regulated by ERK/MAPK signaling.  Given the importance of both of these signaling pathways in melanoma biology, we hypothesize that FAM129B could be an important regulatory nexus between Wnt/β-catenin and ERK/MAPK signaling, although at this point we have been unable to draw a direct connection in patient tumors. Thus, at this point it is difficult to make any conclusions on the role of FAM129B’s role in melanoma treatment without being overly speculative. “I feel that the authors could provide a more in-depth discussion on their findings and place them in the context of the background information provided in the introduction part”We have added some more content to the discussion, and we hope that these changes have improved the manuscript. “How do the authors integrate the beneficial effects of enhanced Wnt responsiveness (this report) and the previously described enhanced tumour cell invasion (ref 20) of FAM129B expressing melanoma cells?” You have raised a very interesting and relevant point regarding the difficulties in broadly generalizing results obtained in cultured cell systems across a disease. Our evaluation of published data (presented in this revision) also shows increased expression of FAM129B in a cohort previously defined as more invasive, and with lower activation of Wnt/β-catenin signaling . These data would suggest that in patients, the effect of FAM129B on melanoma cell invasion may supersede any effects on regulating Wnt/β-catenin signaling. In response to your helpful suggestion, we have added a model which shows how Wnt/β-catenin and ERK/MAPK pathways modulate apoptosis in melanoma cells .  Thank you for your careful reading of the figure legends, we have edited the legend of Figure 2a to clarify that our western blot of FAM129B is for endogenous FAM129B protein levels.We appreciate your thorough reading of our manuscript, as well as the thoughtful questions that you have raised in your review.  We have outlined a response to specific questions below:


Wnt/β-catenin signaling plays a major role in embryonic development and adult stem cell maintenance. Reduced activation of the Wnt/β-catenin pathway underlies neurodegenerative disorders and aberrations in bone formation. Screening of a small molecule compound library with a β-galactosidase fragment complementation assay measuring β-catenin nuclear entry revealed bona fide activators of β-catenin signaling. The compounds stabilized cytoplasmic β-catenin and activated β–catenin-dependent reporter gene activity. Although the mechanism through which the compounds activate β-catenin signaling has yet to be determined, several key regulators of Wnt/β-catenin signaling, including glycogen synthase kinase 3 and Frizzled receptors, were excluded as the molecular target. The compounds displayed remarkable selectivity, as they only induced β-catenin signaling in a human osteosarcoma U2OS cell line and not in a variety of other cell lines examined. Our data indicate that differences in cellular Wnt/β-catenin signaling machinery can be exploited to identify cell type-specific activators of Wnt/β-catenin signaling. Wnt/β-catenin signaling orchestrates embryogenesis and adult stem cell maintenance in mammals Aberrations in Wnt/β-catenin signaling activity are associated with several malignancies We tested 2300 drug-like compounds with activity towards G protein-coupled receptors (GPCR) or kinases for their ability to activate Wnt/β-catenin signaling in a human osteosarcoma U2OS cell line (termed U2OS-EFC) genetically engineered to couple nuclear entry of β-catenin to increases in β-galactosidase complementation At a 10 µM concentration, both Cpd1 and Cpd2 induced transcription of a transiently transfected SuperTOPflash reporter gene in U2OS-EFC cells . We obseBecause Frizzleds are structurally related to GPCRs, we investigated whether Cpd1 and Cpd2 are capable of activating Frizzleds. To assess this, we used recombinant human Dickkopf-1 (rhDkk-1) protein, which recruits LRP5/6 to the membrane protein Kremen, leading to internalization of LRP5/6 Although biochemical profiling indicated that Cpd1 or Cpd2 did not act through inhibition of GSK3 or any other protein kinase, the possibility that the compounds inhibit GSK3 activity by inducing phosphorylation of an N-terminal serine residue (Ser21 in GSK3α and Ser9 in GSK3β) can only be verified in cells. Phosphorylation of GSK3β at Ser9 is catalyzed by protein kinase B (PKB)/Akt, when cells are stimulated with insulin or platelet-derived growth factor (PDGF) 696 structural analogs of Cpd1 and Cpd2 were selected from the Merck research Laboratories compound library and tested in the β-catenin EFC assay. 18 compounds were found to increase β-galactosidase activity at 10 µM and two compounds  and glucocorticoid hormone receptor (GR) signaling. None of these reporter genes was activated by Cpd1, whereas SuperTOPflash reporter gene activity was dose-dependently induced by Cpd1 treatment  and cultured in DMEM F12 containing 10% fetal bovine serum , 100 U/ml penicillin and 100 µg/ml streptomycin . U2OS-Δα-Nuc cells  were maintained in the same medium, supplemented with 150 µg/ml hygromycin (Invitrogen). U2OS-EFC, U2OS-GR and HEK293-EFC cells, the generation of which is described elsewhere Synthetic organic low-molecular weight (LMW) compounds were selected from the Merck Research Laboratories compound collection  based on previously determined activity on various G protein-coupled receptor (GPCR) or protein kinase targets. Recombinant mouse Wnt-3a (rmWnt-3a), recombinant human Wnt inhibitory factor-1 (WIF1) and recombinant human Dickkopf-1 (rhDkk-1) were purchased from R&D Systems . Platelet-derived growth factor (PDGF) was obtained from PeproTech .The β-catenin EFC assay was performed as described previously Luciferase assays on transiently transfected cells were performed as described before Isolation of cytoplasmic fractions and total cell lysates, followed by western blotting, was described previously Isolation of total mRNA and subsequent analysis of transcript levels by qPCR was performed as described previously 50 values) for rhDkk1 and rmWnt-3a dose response curves was calculated from the expected molecular mass of rhDkk-1 and rmWnt-3a (both 41 kDa) using Graphpad Prism 4.0 software. All data are represented as averages ± standard error in the mean (SEM). Statistical significance of observed differences was determined using Student's t-test and indicated in the figures with asterisks (*). P<0.05 was regarded as statistically significant.The concentration at which half-maximal activation of the cellular response was reached  by means of qPCR. Asterisk represent statistically significant differences (p<0.05).(TIF)Click here for additional data file.Figure S2Schematic representation of the mode of action of Cpd1 and Cpd2. A model of Wnt/β-catenin signaling, in which activation of Frizzled receptors by Wnt-3a leads to subsequent phosphorylation of LRP5/6, inhibition of GSK3, β-catenin accumulation, β-catenin nuclear translocation and β-catenin-dependent gene transcription. Cpd1 and Cpd2 activate Wnt/β-catenin signaling at the level of β-catenin accumulation.(TIF)Click here for additional data file.Figure S3Structural analogs of Cpd1 and Cpd2 activate Wnt/β-catenin signaling in U2OS-EFC cells. U2OS-EFC cells were treated for 3 hrs with 10 nM rmWnt-3a or 10 µM of Cpd1-5, before measurement of β-galactosidase activity.(TIF)Click here for additional data file.Figure S4Cpd1 and Cpd2 do not activate Wnt/β-catenin signaling in several commonly used cell lines from different origins. HeLa (A), CHO-k1 (B), Saos-2 (C), UMR106 (D) and U2OS cells derived from ATCC (E) were transiently transfected with TOPflash reporter gene construct and stimulated with 10 µM Cpd1, Cpd2 or CP55940 or 12 nM rmWnt-3a for 5 hrs prior to measurement of luciferase activity.(TIF)Click here for additional data file.Figure S5Cpd1 activates SuperTOPflash, but not CREB-, NFAT- and GR-dependent reporter gene activity in U2OS-Δα-Nuc cells. U2OS-Δα-Nuc cells were transiently transfected with vectors encoding luciferase under the transcriptional control of (A) β-catenin (SuperTOPflash), (B) CREB (21xCRE-luc), (C) NFAT/Ca2+ (NFAT-luc) and (D) GR (MMTV-luc). Cells were stimulated with ascending concentrations of Cpd1 and reference agonists: (A) 10 nM rmWnt-3a, (B) 10 µM isoproterenol, an agonist for endogenously expressed Gs-coupled β2 adrenergic receptors, (C) a combination of 100 nM phorbol 12-myristate 13-acetate (PMA) and 100 nM thapsigargin (Thaps), which activate protein kinase C and Ca2+-signaling, respectively, and (D) 1 µM of the GR agonist dexamethasone (Dex).(TIF)Click here for additional data file.Figure S6Cpd1 does not activate β-galactosidase activity in a CHO cell line genetically engineered to couple recruitment of the scaffolding protein β-arrestin2 to the human parathyroid hormone receptor 1 (hPTH1R). CHO-PTH1R cells were stimulated with increasing concentrations of Cpd1 or with 100 nM human PTH1-34 for 90 min, followed by measurement of β-galactosidase activity.(TIF)Click here for additional data file.Figure S7The increase in β-galactosidase activity in U2OS-EFC cells following treatment with Cpd1 has a profile that is similar to that observed for U2OS-EFC cells treated with LiCl. U2OS-EFC cells were treated with 10 µM Cpd1 or 30 mM LiCl for several time periods before measurement of β-galactosidase activity.(TIF)Click here for additional data file.


TIG1) is a putative tumor suppressor gene. We have recently demonstrated both TIG1A and TIG1B isoforms inhibited cell growth and induced the expression of G protein-coupled receptor kinase 5 (GRK5) in colon cancer cells. Because elevated prostaglandin E2 (PGE2) signaling plays a significant role in colorectal carcinogenesis, the objective of this study was to explore the effect of TIG1 on PGE2-induced cellular proliferation and signaling in colon cancer cells.The tazarotene-induced gene 1 (HCT116 cells as well as TIG1A and TIG1B stable cells established from HCT116 colon cancer cells using the GeneSwitch system were used. TIG1 isoform expression was induced by mifepristone treatment in stable cells. Cell growth was determined using the WST-1 cell proliferation assay. Activation of β-catenin/TCF and cyclic adenosine monophosphate (cAMP)/CREB signaling pathways were determined using luciferase reporter assays. Expression and subcellular distribution of β-catenin were analyzed using Western blot and confocal microscope. Levels of cAMP were measured using an enzyme immunoassay. RNA interference was used to examine the effects of TIG1- and GRK5-mediated changes.PGE2-stimulated cell growth was reduced in inducible TIG1A- and TIG1B-stable HCT116 cells. GRK5 expression was upregulated by both TIG1A and TIG1B isoforms, and its expression suppressed PGE2-stimulated HCT116 cell growth. GRK5, TIG1A, and TIG1B expression significantly inhibited PGE2-stimulated β-catenin/TCF and cAMP signaling pathway reporters and cAMP. Also, PGE2-stimulated nuclear localization of β-catenin was inhibited by expression of TIG1A and TIG1B, which was ameliorated by both TIG1 and GRK5 siRNAs.TIG1 suppressed PGE2-stimulated Wnt and cAMP signaling pathways in colon cancer cells through GRK5. TIG1) gene, also known as retinoic acid receptor responder 1 (RARRES1) gene  × 100%. All experiments were performed in triplicate.Control and TIG1-inducible HCT116 cells  containing 20 μg/mL aprotinin and 20 μg/mL phenylmethylsulfonyl fluoride and 2 mM NaF and 1 mM Na3VO4. Proteins (5 to 100 μg) were separated using 10-12% polyacrylamide gels containing sodium dodecyl sulfate and were then transferred to polyvinylidene difluoride membranes. After blocking, the membranes were incubated with anti-V5 (Invitrogen), anti-myc (Invitrogen), anti-GRK5 , anti-β-catenin , anti-lamin B1 (Invitrogen), anti-glyceraldehyde 3-phosphate dehydrogenase , or anti-actin (Sigma) antibodies for 12 h at 4°C. The appropriate horseradish peroxidase-conjugated secondary antibodies  was then added at room temperature for 1 h. Specific protein bands were developed using the Amersham ECL . Relative protein expression was quantified following normalization to the housekeeping gene protein levels.Total cytosol extracts were prepared in lysis buffer  according to the manufacturer's instructions with the addition of protease and phosphatase inhibitors to all buffers. Nuclear (10 μg) and cytosolic (25 μg) extracts were analyzed using Western blot analysis. Cytosolic β-catenin levels were first normalized to GAPDH levels, and nuclear β-catenin levels were normalized to lamin B1 levels. Cytosolic β-catenin was then normalized to cytosolic β-catenin protein levels in the respective cells or control stable cells without PGE2 treatment. Similarly, nuclear β-catenin levels were then normalized to nuclear β-catenin protein levels in the respective cells or control stable cells without PGE2 treatment for the siRNA study. Finally, relative levels of nuclear β-catenin relative to that of cytosolic β-catenin were calculated, and the level of nuclear β-catenin in the respective cells or in control stable cells without PGE2-treatment was designated as 1.0.5) were plated in six-well plates in growth medium for 24 h. After transient transfection with 500 ng plasmids, the cells were serum starved for 16 h and stimulated with 10 nM PGE2 for 24 h at 37°C. Alternatively, inducible TIG1A, TIG1B, or control stable cells were plated overnight, transfected with 500 ng reporter plasmid, incubated with 5 nM MFP for 24 h, serum starved for 16 h, and incubated with PGE2 (10 nM) for 24 h. To analyze the effect of GRK5 or TIG1 knockdown on reporter activity, inducible or control stable cells were cotransfected with siRNAs and reporter plasmids followed by serum starvation and then stimulation with 5 nM MFP for 24 h. MFP was present during the subsequent serum starvation and PGE2 treatment.TOPFLASH  and CREB reporter  activities were assessed in transiently transfected HCT116 cells. HCT116 cells . Luciferase activity was measured using the luciferase assay kit (Stratagene) and a multi-functional microplate reader . Relative luciferase activity of each sample was determined after normalizing for the protein concentration of each lysate. All experiments were performed in triplicate.5) were plated on poly-L-lysine-coated coverslips in 35-mm dishes in growth medium. Cells were treated with 5 nM MFP for 24 h, maintained in serum-free medium for 16 h, treated with 10 nM PGE2 for 30 min, washed twice with PBS, and fixed with 4% paraformaldehyde. Alternatively, cells were transfected with siRNAs prior to MFP treatment, serum starvation, and PGE2 treatment. MFP was present during the serum starvation and PGE2 treatment. Cells were permeabilized in 0.1% Triton X-100/PBS for 15 min. After washing, cells were blocked in PBS containing 2% bovine serum albumin for 1 h at room temperature, incubated with anti-β-catenin antibody at 37°C for 2 h followed by the Alexa Fluor@ 488 goat anti-mouse IgG for 1 h at room temperature, stained with 1 μg/mL 4'6-diamidino-2-phenylindole (DAPI), and analyzed with a laser scanning confocal microscope . Percentage of cells expressing nuclear β-catenin was determined by randomly analyzing 250 to 300 cells from each treatment group.Stable cells  according the manufacturer's instructions.t-test. P-values less than 0.05 were considered statistically significant.Numerical data are shown as mean ± standard deviation (SD) of triplicates from each independent experiment. Cell proliferation and luciferase activities were analyzed using the student's In the COX-2-negative HCT116 cells, TIG1A and TIG1B expression was markedly induced after the respective stable cells were incubated with 5 nM MFP for 24 and 48 h  treatment for 48 h significantly stimulated cell proliferation, and the effect was not altered by co-incubation with 5 nM MFP Figure . HoweverTOPFLASH and CREB reporter assays were first used to investigate the effects of TIG1 expression on PGE2-stimulated Wnt and PKA signaling pathways by measuring β-catenin/TCF- and cAMP/CREB-mediated reporter activities, respectively. PGE2 treatment significantly stimulated TOPFLASH and CREB reporter activities in control HCT116 stable cells Figure . HoweverThe effects of TIG1 on PGE2-stimulated β-catenin nuclear localization in HCT116 cells were then determined by Western blot analysis of cytosolic and nuclear extracts. After exposure to 10 nM PGE2 for 5 min to 1 h, the greatest nuclear β-catenin accumulation as determined by immunofluorescent analysis was evident at 30 min in control stable cells (data not shown). The majority of TIG1 isoforms were detected in the cytosolic fractions Figure . Levels Thus, upon induced expression of both TIG1A and TIG1B, suppression of both PGE2-stimulated TOPFLASH reporter activities and nuclear β-catenin localization in MFP-treated stable HCT116 cells was observed. PGE2-stimulated CREB reporter activities were also suppressed by TIG1B, but to a lesser extent.To investigate the effects of GRK5 on PGE2-mediated signal pathways, GRK5 expression vector was constructed. Expression of the GRK5 fusion protein with an expected molecular weight of 67 kDa was observed in HCT116 cells Figure . In vectAs shown in Figure Similar experiments in stable TIG1A expressing cells revealed a partial knockdown of TIG1A (65.1%) and GRK5 (50%) protein expression using the respective siRNAs Figure . Levels The effects of GRK5 and TIG1 knockdown on PGE2-stimulated β-catenin nuclear localization were evaluated in MFP-treated stable cells. In MFP-treated control stable cells transfected with control siRNA, levels of nuclear β-catenin were designated as 1.0-fold in the absence of PGE2 treatment; treatment with 10 nM PGE2 for 30 min increased nuclear β-catenin to 1.4-fold Figure . In stabUsing siRNA, TIG1A-mediated suppression of TOPFLASH reporter activity and nuclear β-catenin localization was alleviated by both TIG1 and GRK5 siRNAs, whereas GRK5-mediated suppression of TOPFLASH reporter activity was reversed by GRK5, but not TIG1 siRNA. Similar, but less potent, effects were observed for CREB reporter activity.The effects of TIG1 on PGE2-stimulated cAMP/PKA signaling were analyzed by measuring the levels of cAMP. PGE2 stimulation for 5 min significantly increased cAMP levels in control stable HCT116 cells, but not in TIG1A- or TIG1B-expressing cells. Levels of PGE2-stimulated cAMP were significantly decreased by 52.2 and 42.8% in TIG1A- and TIG1B-expressing cells, respectively  and stimulating Wnt/LRP6 signaling . TherefoTIG1 gene expression [There are several study limitations that warrant consideration. Firstly, HCT116 cells harbor a somatic β-catenin mutation rendering it resistant to GSK-3β-dependent phosphorylation and degradation ,33. Nevepression ,9. Loss pression  and an ipression  are relaIn conclusion, both TIG1A and TIG1B suppressed PGE2-stimulated colon cancer cell proliferation, β-catenin nuclear localization, β-catenin/TCF transactivation, and cAMP/CREB signaling through expression of GRK5. The suppression of β-catenin/TCF transactivation and β-catenin nuclear localization was mediated through GRK5. The results suggest an inhibitory role of TIG1 on PGE2-mediated colorectal carcinogenesis at least in part, through upregulation of GRK5 expression.APC: adenomatous polyposis coli; AGBL2: ATP/GTP binding protein-like 2; CREB: cAMP response element binding protein; CRE: cAMP response element; cAMP: cyclic adenosine monophosphate; COX-2: cyclooxygenase-2; DAPI: 4'6-diamidino-2-phenylindole; GPCR: G protein-coupled receptor; GRKs: G protein-coupled receptor kinases; GSK-3β: Glycogen synthase kinase 3β; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; LRP6: lipoprotein receptor-related protein 6; MFP: mifepristone; PI3K: phosphatidylinositol 3-kinase; PBS: phosphate buffered saline; PGE2: prostaglandin E2; RARRES1: retinoic acid receptor responder 1; siRNA: small interfering RNAs; SD: standard deviation; TIG1: tazarotene-induced gene 1; TCF-7: transcription factor 7.The authors declare that they have no competing interests.F-MT performed the majority of the experiments, contributed to the experimental design, and drafted the manuscript. C-CW, R-YS and C-HW contributed to experimental design and data discussion. S-YJ designed and supervised the experiments, assisted in the writing of and proofed the manuscript. All authors read and approved the final draft of the manuscript.Effects of TIG1A and TIG1B expression on PGE2-induced SW620 cell growth. Control, TIG1A and TIG1B inducible stable cells established from SW620 colon cancer cells using the GeneSwitch system [P < 0.05.h system  were plaClick here for fileGRK5 suppressed PGE2-stimulated TOPFLESH and CREB reporter activities in EP2-transfected HCT116 cells. HCT116 cells were transiently transfected with 250 ng of TOPFLASH or CREB reporter plasmid along with 100 ng of indicated control vector or EP2 TrucClone™ cDNA  and 50 to 150 ng of GRK5 expression vectors for 24 h. Cells were serum starved for 16 h and then incubated with or without 10 nM PGE2 for 24 h. Reporter activities were measured as described in the Materials and Methods. Representative results were expressed as means ± SD from triplicate samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001.Click here for fileTIG1 and GRK5 siRNAs increased PGE2-stimulated nuclear β-catenin accumulation in TIG1A-expressing cells. Control or TIG1A stable cells were transfected with the indicated siRNA and then incubated with 5 nM MFP for 24 h. Cells were serum starved for 16 h and then stimulated with 10 nM PGE2 for 30 min. MFP was present during serum starvation and PGE2 treatment. β-catenin localization (green) and nuclei (blue) were analyzed using a laser scanning confocal microscope. Bars, 10 μm. Arrows indicate cells expressing nuclear β-catenin.Click here for fileEffects of PGE2 on nuclear β-catenin localization in control siRNA transfected TIG1A-expressing cells. TIG1A stable cells were transfected with control siRNA and then incubated with 5 nM MFP for 24 h, serum starved for 16 h, and treated with or without 10 nM PGE2 for 30 min. MFP was present during serum starvation and PGE2 treatment. Nuclear and cytosolic fractions were prepared, and subcellular distribution of nuclear and cytosolic β-catenin was determined by Western blot analysis. Normalization in the levels of nuclear and cytosolic β-catenin was described in the Materials and Methods, and relative levels of nuclear β-catenin in cells without PGE2-treatment was designated as 1.0.Click here for file


Conversely, a decreased T cell recruitment wasassociated with I249T280 haplotype in the controls (64%). The significant higher presence ofthe variant allele I249 in homo- and heterozygosis, found in controls (91%) and in AMI survivors(94%), with respect to the patients who died of AMI (48%), showed the relevance of this polymorphismboth in the onset and outcome of acute myocardial infarction. The presence of CX3CR1polymorphisms could influence the incidence and the outcome of acute myocardial infarction,altering the inflammation of the whole coronary tree by the impaired recruitment of Th1 polarized subpopulation in the coronary plaque.Fractalkine is a proinflammatory chemokine that participates in atherosclerotic processmediating the interactions of vascular cells and leukocytes and selective recruitment ofTh1 lymphocytes, through interaction with CX3CR1 receptor. The polymorphism of thefractalkine receptor 280M-containing haplotype, which codifies for a receptor with minorexpression and with a reduced binding capability, represents a novel protective factor ofatherosclerotic disease. We investigated the association among CX3CR1 genotype, theinflammatory infiltrate subpopulations recruited in the plaque, and the in situ expression offractalkine and its receptor, in patients who died of myocardial infarction (AMI) compared withsubjects who died of noncardiac causes. Patients with nonlethal AMI (AMI survivors)were also investigated to correlate the CX3CR1 polymorphisms and the incidence of lethal AMI.A strong T cells infiltrate was found in infarct related artery (IRA) plaques of AMI patientspresenting the V Genetic factor appears to be important determinant of cardiovascular disease. In particular, specific genetic polymorphisms that modulate blood pressure, coagulation, and lipid metabolism have been identified to be associated with risk of coronary artery disease. However, none of these associations has identified the precise molecular basis of pathological accumulation of leukocytes in vessel wall that characterized the onset of acute myocardial infarction.Acute myocardial infarction (AMI) in fact is related to a diffuse active inflammation of the coronary tree associated with rupture of one of the multiple vulnerable plaques.Unlike stable plaques characterized by a chronic infiltrate of macrophages, vulnerable plaques display an inflammatory infiltrate composed by T cells activated towards a Th1 pathway, and whose recruitment is regulated by chemokines, cytokines, and their receptors .In particular, chemokines are part of effector and amplification mechanisms of polarized Th1 or Th2 mediated immune responses; therefore, their receptors may serve as Th1 versus Th2 marker and lead to the selective modulation of T cell dependent immunity. Hence, the receptor expression and the chemokines release are closely linked to T cell polarization and activation.Fractalkine CX3CL1) is a proinflammatory chemokine that induces chemotaxis of circulating monocytes and selective recruitment of Th1 lymphocytes through interaction with its ligand, the CX3CR1 receptor, that is highly expressed on Th1 activated T cells CL1 is a . It has Studies on CX3CR1 receptor knock out animal models and on human coronaries with native atherosclerosis point out the important role of fractalkine in atherogenesis and plaque progression , 13.Recent data demonstrated the enhanced expression of CXCL1 and its receptor (CX3CR1) in coronary artery disease that could be downmodulated by statin therapy . These d249M280 haplotype codifies for a chemokine receptor variant that has impaired adhesive function, low binding activity, and low recruitment efficiency of T cells within the plaque. On the contrary, the CX3CR1 V249T280 haplotype, characterized by high expression and binding efficiency, is associated with high risk of CAD and could be related to plaque vulnerability and rupture.Epidemiological studies have proved the existence of two common nonsynonymous single-nucleotide polymorphisms of CX3CR1 receptor gene (V249I and T280M). The polymorphisms of the fractalkine receptor, that codify for a molecule with minor expression and with a reduced binding capability, have been proposed as a novel protective marker for coronary disease. In particular, the CX3CR1 I249 allele in homo- and heterozygote conditions has been associated with a marked reduction in risk of acute coronary events –17. In aAll these data suggest that the CX3CR1 V249 and T280 polymorphisms could amplify the Th1 response triggering plaque vulnerability and rupture. Moreover, the higher expression and binding activity of this receptor could influence the atherogenesis in patients with artery disease and the incidence and the outcome of the myocardial infarction. Little information is available regarding the risk factors predicting the subsequent lethal cardiac events after acute myocardial infarction (AMI). In fact, despite the use of advanced medical and invasive procedures, the risk adjusted 90-day mortality rates in patients with myocardial infarction are up to 20–24%. Recent data pointed out the relevant role of diffuse inflammatory process. We have recently demonstrated, in patients who died of a first AMI, the presence of a strongly activated T cell infiltrate in the entire coronary tree and in the peri-infarctual and in the remote unaffected myocardial regions underlying both coronary and myocardial vulnerability , 19. In Therefore, we evaluated the inflammatory burden and the in situ expression of fractalkine and its receptor in coronary plaques of patients died of myocardial infarction (AMI) as compared to subjects died of noncardiac causes in order to focus if the presence of I249/M280 haplotype correlates with an impaired Th1 cell recruitment in the plaque.We further evaluate if the CX3CR1 polymorphisms could influence the outcome of acute myocardial infarction. At this purpose, we examined the of CX3CR1 polymorphisms presence in individuals who died of noncardiac causes, in a cohort of patients that survived at acute myocardial infarction (AMI survivors) compared with patients died of AMI.We observed 25 patients  died of acute myocardial infarction (AMI group) and 22 age-matched patients  died of noncardiac causes and without clinical cardiac history (NCC group). Further, 16 patients  who survived at a previous myocardial infarction (MI) suitable for surgical ventricular restoration have been analyzed (SVR group). Clinical, angiographic and surgical characteristics are listed in The main indications for surgery of patients of SVR group were symptoms of heart failure, angina, and/or a combination of the two. Four patients of this group (25%) had associated mitral valve repair for a grade 3+/4+ of mitral regurgitation. The surgical technique has been previously described . Mitral Patients with chronic inflammatory diseases or tumors were excluded from the study to avoid bias due to immunological changes. All autopsies were performed within 12–24 hours of death. At the autopsy, in AMI and NCC, the 4 major epicardial coronary arteries  were dissected from each heart, 10% buffered formalin fixed, cut transversely at 5 mm intervals, decalcified if necessary, paraffin embedded, and serially sectioned for morphological and immunohistochemical studies. The hearts were transversally cut at 1 cm intervals from the apex to the base. The myocardium was examined for the presence and extent of infarction. The location of myocardial infarction was detected by immersion of a cross-section of the heart in a solution of triphenyltetrazolium chloride . In addition, in all cases, another cross-section of the heart was processed for histological examination, and the infarction was confirmed by light microscopy.In SVR patients, LV myocardial samples were obtained at the time of surgical operation. The tissue was taken from the transition zone between MI and the remote, normal region. All tissues were fixed in 10% formalin and embedded in paraffin.Coronary sections from the two groups, H&E and pentachrome stained, were examined by light microscopy. Culprit plaques were defined as lesions showing either plaque rupture or erosion with superimposed thrombus. The coronary artery showing the culprit lesion was defined as infarct-related artery (IRA). In AMI were examined: the culprit segment in the IRA, including the thrombotic lesion, 1.5 cm long, a segment 1.5 cm long in which maximum of stenosis was present in the non-IRA coronary. In NCC, a segment of 1.5 cm long in which maximum of stenosis was present was selected. All coronary segments were cut transversely at 5 mm intervals. A total of 216 arterial segments were paraffin embedded and serially sectioned: 150 from the AMI group (75 from IRA and 75 from non-IRA coronaries) and 66 from NCC group, respectively.μm thick sections were cut from paraffin blocks and mounted on SuperFrost Plus slides . α-smooth actin, CD68, CD3 , fractalkine , and CX3CR1  primary monoclonal antibodies were used. CD68  and CD3 (DAB detected) IHC was used to quantify the inflammatory infiltrate. For each antibody, positive cells, as a mean of 10 randomly selected fields per section, were counted at a magnification of 40X in the plaque shoulder region and in the cap itself.Serial 3 X-, Y-, and Z-axis.In order to characterize the cell subpopulations that express CX3CR1, we performed a triple immunostaining for confocal microscopy. Paraffin sections of coronary plaques were first incubated with anti-CD3 MoAb , rinsed, and then incubated with biotinylated mouse IgG; Fluorescence was obtained by incubating the sections with a streptavidin-coumarin fluorochrome. A second reaction with primary antibodies to CX3CR1  was performed. Fluorescence was obtained with a conjugate streptavidin-fluorescein conjugate (FITC). A third immunostaining was performed by incubating the sections with anti-IFN gamma monoclonal antibody . Sections were incubated with tyramide signal amplification biotin system . Fluorescence was obtained with a streptavidin-Texas Red fluorescent conjugate. Control sections were incubated with a mixture of irrelevant monoclonal reagents having a similar isotype. Images were acquired by means of Noran confocal microscope at 60X/1.4 NA immersion oil lens. Three D stacks were acquired at a resolution of 0.1 micron in μL of Buffer AE.DNA was extracted from formalin fixed and paraffin embedded heart of the subjects described above. Paraffin was removed with xylene for 5 min at room temperature. Samples were centrifuged at full speed for 5 min at RT. Supernatant was removed, and 1.2 mL of 100% ethanol was added to remove residual xylene. The DNA was extracted with the QIAamp DNA kit  and resuspended in 50 μL of PCR reaction was digested, respectively, with Acl I  or BsbmB1  in 20 μL of reaction as previously described [The DNA quality was controlled by a genomic polymerase reaction assay (PCR) using the primer set for BAK gene. The following two polymorphisms have been analyzed: I249, a guanidine to adenine substitution at nucleotide 745 of the open reading frame changing valine at position 249 to isoleucine, and M280, a cytosine to thymidine substitution at nucleotide 839 changing a threonine at position 280. The PCR product was analyzed in 2% agarose gel stained with ethidium bromide. CX3CR1 gene T280M and V249I mutations were identified after amplification of 311 base pairs (bp) . The T280M and V429I were detected on the same amplified fragment. Amplification reactions were performed using 35 cycles of 95°C, 69°C, and 72°C for 30 seconds each, preceded by a single cycle of 95°C for 3 minutes and followed by a single cycle of 72°C for 10 minutes. In order to type the alleles at codons 249 and 280, 5 t-test was utilized to assess intergroup differences for continuous variables. The X2 test was used to establish differences in the distribution between categorical variables. The statistical significance of different polymorphisms present in the single groups was identified by Fisher's exact test. A P value of <0.05 was considered statistically significant.Statistical analysis of the data was performed with SPSS for Windows . Unpaired Using logistic regression analysis (SPSS 13.0), the association of each polymorphism with the cardiovascular events  was adjusted for age, gender, smoking cigarette, hypertension, diabetes mellitus, and hyperlipidemia.The examination of the whole coronary tree of the AMI group demonstrated the presence of multiple complicated atherosclerotic plaques with a thin fibrous cap and a high inflammatory infiltrate; moreover, in the coronary branch supplying the infarct area (IRA), we found a disrupted/thrombotic plaque (culprit plaque). Conversely, the coronary trees of the control group (NCC) showed atherosclerotic lesions with a thick fibrous cap and a scanty inflammatory infiltrate. No plaque disruption or erosion or thrombosis has been observed. Clinical, autoptic, and surgical patient characteristics are showed in α-actin). A quantitative analysis demonstrated a significant (P < 0.01) increase in the inflammatory cells per area (CD3 and CD68 positive) in AMI plaques (51.4 ± 2.5%) as compared with the plaques of the NCC group (22.4 ± 1.9%). T lymphocytes represented the 18.5 ± 2.3% of the plaque cells in AMI and the 6.4 ± 1.8% of cells in the NCC, whereas macrophages were 32.9 ± 3.2% in the AMI and 16.0 ± 1.8% in the NCC (γ was assessed in culprit lesions by immunofluorescence staining (coronary artery from AMI group) with confocal microscopy , macrophages (CD68), and smooth muscle cells  involved in plaque inflammation and destabilization correlated with the presence of Th1 polarized T cells (CXCR1 expressing cells) recruited in the plaque by the interaction of fractalkine and the CX3CR1 receptor on the T cell membrane.The immunohistochemical studies demonstrated that endothelial cells in plaques of AMI patients gave rise to a diffuse positive reaction for fractalkine. Smooth muscle cells were focally positive, while lymphocytes and macrophages were not. Conversely, in the plaques of NCC group, all the cell types were almost negative for fractalkine .The fractalkine receptor (CX3CR1) was strongly and diffusely expressed in the cell membrane in T lymphocytes of AMI plaques  as compaγ expression confirmed that T lymphocytes expressed IFN-γ .In the coronary, plaques from control group, T lymphocytes showed a lower level of CX3CR1 positivity (18% of T lymphocytes were positive) ; in thisγ production in the plaque but a large number of B cells. In P < 0.01), confirming the Th1 polarized subpopulation recruitment in the plaque.It has been previously demonstrated that CX3CR1 genotypes influence the fractalkine receptor binding affinity and therefore Th1 lymphocytes recruitment. In order to correlate the presence of activated Th1 lymphocytes with CX3CR1 genotype, patients were genotyped for the V249I and T280M polymorphisms of CXCR1 gene. DNA extracted from myocardial formalin fixed and paraffin embedded sections was analyzed by polymerase chain reaction. The genotype was determined on the amplicon of 311 bp after digestion with endonucleases specific for each polymorphism. The analysis of CX3CR1 V249I and T280M polymorphisms demonstrated that 13 out of 25 patients who died of infarction (52%) were homozygous for the V249 allele and 21 out of 25 (84%) were homozygous for the T280 allele . Moreoven = 16), in order to compare the presence of the allele variant with the occurrence and the outcome of the acute event. The genotype analysis has been extended to a cohort of individuals survived at a first myocardial infarction (We found that the 52% of AMI patients (13/25) displayed the V249 allele in homozygosis, as compared with the 6% of the AMI survivors (1/16) and the 9% of controls (2/22) .P = 0.001) but not with all the major risk factors.The multivariate analysis of each V249I polymorphism, adjusted for clinical data in the three study groups, showed that the V/V249 haplotype was significantly associated only with the fatal AMI  , as compThe analysis of T280M polymorphism displayed the presence of the M280 variant allele in homozygosis in the 4% of controls (1/22), as compared with AMI and survivors patients, which showed no presence of this allele in homozygosis. The presence of M280 allele in homozygosis was always associated with the I249 allele.In summary, the significant higher presence of the variant allele I249 in homo- and heterozygosis found in controls (91%) and in AMI survivors (94%), with respect to the patients died of AMI (48%), showed the relevance of this polymorphism both in the onset and outcome of acute myocardial infarction, confirming the correlation between CX3CR1 genotype and prognosis of the acute event.Epidemiological and clinical studies have shown that the CX3CR1 receptor polymorphisms are linked with the risk of coronary artery diseases , 15.In particular, the presence of CX3CR1 V249 and T280 single nucleotide polymorphisms codify for a fractalkine receptor that is overexpressed by activated inflammatory cells and with higher binding affinity for fractalkine, its natural ligand , 20. The genotyping analysis of the subjects enrolled in the present study demonstrated for the first time a direct correlation between CX3CR1 V249I and T280M polymorphisms and the occurrence of acute fatal myocardial infarction. A striking link between genotype and prognosis of the acute event was suggested by the observation of AMI survivors genotypes. Notably, the in situ CX3CR1 high expression in AMI patients correlates with the presence of V249 polymorphism in homo- or heterozygosis. Conversely, the genotype analysis of controls (with no CVD history) demonstrated the presence of I249 polymorphism in homozygosis.Indeed, CX3CR1 V249/T280 polymorphisms in homozygosis or heterozygosis have been demonstrated in 92% of the AMI patients analyzed (mean age: 70 years old). In particular, 52% of the AMI patients examined were homozygous for CX3CR1 V249, whereas the analysis of the genotype of the control subjects (mean age: 73.7 years old) demonstrated the presence of CX3CR1 V249 polymorphism in homozygosis only in the 9% of cases. The findings of the high percentage of I/I249 (36%) and M/M280 (4%) in subjects died of no cardiac causes suggest a possible link between CX3CR1 polymorphism, T cell recruitment, and acute event. Moreover, the genotype observations on survivors group suggest that the presence of I249 allele is relevant not only for the occurrence but also in the outcome of the acute event.There is evidence of the influence of CX3CR1 variable expression and function on T cells recruitment efficiency , 21, 22.−/− background [Studies on CX3CR1 null mice, crossed into apoE ckground , demonstIn present work, we also show a close association between CX3CR1 genotypes and coronary plaques T cell inflammation, that is, a relevant character of vulnerable plaques according to data previously published .Indeed, coronary plaques of patients with CX3CR1 I249 allele displayed a weaker T cell inflammation as compared to individuals homozygous for the CX3CR1 V249 allele.In particular, we show that CX3CR1 is highly expressed by the majority of T cells infiltrating IRA plaques of 22 out of 25 AMI patients.It is worth noting that the two patients showing a low expression of CX3CR1 were CX3CR1 I249 homozygou and their plaques were infiltrated by B lymphocytes, suggesting a different pathogenesis for AMI.Conversely, immunostaining for CX3CR1 was negative or faint positive in plaques of controls, where the T lymphocytes infiltrate was lower as compared with AMI patients.Therefore, it can be assumed that the high expression of CX3CR1 together with the in situ expression of fractalkine (CX3CL1) correlates with the increased recruitment of T lymphocytes deeply infiltrating in the plaques of the AMI patients.The special feature of fractalkine among other members of the chemokine family is due to the fact that it is expressed both as soluble and membrane-bound form on the surface of inflamed endothelium. Interestingly, whereas soluble fractalkine (CX3CL1) was reported to recruit lymphocytes and monocytes, immobilized forms of CX3CL1 have been shown to directly mediate the rapid capture and firm adhesion of leukocytes expressing its receptor CX3CR1 under physiological flow conditions . The dirThe coexpression of CD3, IFN-gamma, and CX3CR1 on T cell population found in AMI plaques compared to controls demonstrates the in situ activation of inflammatory cells pointing out the probable involvement of a recent immune Th1 response in plaque destabilization and rupture. Moreover, the data obtained suggest that the susceptibility to vascular inflammation is correlated with the amplified recruitment of Th1 polarized cells expressing the high or low affinity CX3CR1 receptor. The findings revealing CX3CR1 expression by 80% of T cell population along with the presence of fractalkine in IRA plaques suggest that these two events are both likely to play a major role in determining plaque vulnerability by orchestrating Th1 cells recruitment. In agreement with previous clinical and epidemiological studies, our data demonstrate a close association between V249I and T280M polymorphisms, coronary plaque vulnerability, and AMI, confirming that the subjects with I249 allele display a low level of T cell inflammation and a reduced risk of developing vulnerable plaques and they have a decreased incidence of acute events (probably due to a lower CX3CR1 binding affinity) . The comIn conclusion, CX3CR1 genotype could be considered as a new marker to stratify the population according to fatal AMI risk. Moreover, CX3CR1 is likely to amplify the Th1 immune response, directly related to plaque vulnerability, irrespectively of the causal agents (i.e. ox-LDL or Chlamydia), thus, standing out as a potential target in fatal AMI prevention.


Hepatocellular carcinoma (HCC), a major cause of cancer-related death in Southeast Asia, is frequently associated with hepatitis B virus (HBV) infection. HBV X protein (HBx), encoded by a viral non-structural gene, is a multifunctional regulator in HBV-associated tumor development. We investigated novel signaling pathways underlying HBx-induced liver tumorigenesis and found that the signaling pathway involving IκB kinase β (IKKβ), tuberous sclerosis complex 1 (TSC1), and mammalian target of rapamycin (mTOR) downstream effector S6 kinase (S6K1), was upregulated when HBx was overexpressed in hepatoma cells. HBx-induced S6K1 activation was reversed by IKKβ inhibitor Bay 11-7082 or silencing IKKβ expression using siRNA. HBx upregulated cell proliferation and vascular endothelial growth factor (VEGF) production, and these HBx-upregulated phenotypes were abolished by treatment with IKKβ inhibitor Bay 11-7082 or mTOR inhibitor rapamycin. The association of HBx-modulated IKKβ/mTOR/S6K1 signaling with liver tumorigenesis was verified in a HBx transgenic mouse model in which pIKKβ, pS6K1, and VEGF expression was found to be higher in cancerous than non-cancerous liver tissues. Furthermore, we also found that pIKKβ levels were strongly correlated with pTSC1 and pS6K1 levels in HBV-associated hepatoma tissue specimens taken from 95 patients, and that higher pIKKβ, pTSC1, and pS6K1 levels were correlated with a poor prognosis in these patients. Taken together, our findings demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ, which is crucially linked to HBV-associated HCC development. Furthermore, we investigated the status of IKKβ/TSC1/mTOR signaling in specimens from HBV-associated human hepatomas, and analyzed the relationship between the status of IKKβ/TSC1/mTOR signaling and the prognosis of HCC patients. We conclude that IKKβ activates mTOR signaling through TSC1 suppression to contribute to one crucial mechanism underlying HBx-dependent pathogenesis of HCC.Hepatocellular carcinoma (HCC), which occurs frequently in Southeast Asia, is one of the most important causes of cancer-related death in the world 5′–AAGCTTGCTGCTCGGGTGTGCTGCCAA–3′ and Reverse 5′–GGTACCGG CAGAGGTGAAAAAGTTGCA–3′. The sequence of the HBx cDNA was confirmed by sequencing analysis. The expression vectors payw1.2WT and payw*7 The pcDNA6.0-HBx plasmid was constructed by cloning the cDNA product of the HBx gene into the pcDNA6.0 expression vector. The cDNA of the HBx gene was obtained by preparing RNA from serum of an HBV (+) patient followed by a reverse transcription-polymerase chain reaction (RT-PCR) using reverse transcriptase , oligo(dT) primers, and HBx primers: Forward 2 incubator with Dulbecco’s modified Eagle’s/F12 medium plus 10% fetal bovine serum.Hep3Bx and HepG2x cells were derivatives of human hepatoma Hep 3B and Hep G2 cells (both were from ATCC), respectively, stably expressing the HBx gene, and were established by transfecting Hep3B and HepG2 cells with pcDNA6.0-HBx using Lipofectamine (Invitrogen) followed by drug selection The primary antibodies used in this study were anti-TSC1 , anti-phosphorylated S6 kinase (T389) , anti-S6 kinase , anti-phosphorylated IKKβ (S181) , anti-IKKβ , anti-HBx , anti-VEGF-A , anti-CD31 , and anti-actin . The rabbit polyclonal antibody against the phospho-S511 of TSC1 was a kind gift from Dr Mien-Chie Hung . The expression of IKKβ, pIKKβ (S181), TSC1, pTSC1 (S511), S6K1 or pS6K1 (T389) was detected in cell lysates prepared from cells lysed as described previously BAY 11-7082 and rapamycin were purchased from Calbiochem , and recombinant human TNF-α was from Roche Applied Sciences . Hep3B, Hep3Bx, HepG2, and HepG2x cells were grown in complete medium and exposed to 20 µg/ml TNF-α  with or without pretreatment of 40 µM BAY 11-7082 for 45 min  or 100 nM Rapamycin  for 3 h.The MTT assay was used to measure cell proliferation and viability in 5,000 cells seeded onto 96-well plates, treated with TNF-α with or without BAY 11-7082 or Rapamycin for 24 h. The amount of VEGF protein in the culture supernatants was assessed using ELISA  according to the manufacturer’s instructions. IKKβ siRNAs  and control siRNAs  were transfected into cells using Lipofectamine to knock down IKKβ expression.5′–AAGCTTGCTGCTCGGGTGTGCTGCCAA–3′ and Reverse 5′–GGTACCGG CAGAGGTGAAAAAGTTGCA–3′; VEGFA Forward 5′–CATGAACTTTCTGC TGTCTTGG–3′ and Reverse 5′–CATTTGTTGTGCTGTAGGAAGC–3′; GAPDH Forward 5′–TGAAGGTCGGAGTCAACGGATTTGGT–3′ and Reverse 5′–CATG TGGGCCATGAGGTCCACCAC–3′. For real time RT-PCR reactions, 1 µg of total RNA from each sample were used in the RT reaction . The TaqMan gene expression real time PCR assays  were used to assess the mRNA expression levels of the endogenous VEGFA and GAPDH . Expression analysis was done in triplicate for each sample. In each run, the endogenous control gene (GAPDH) and one no-template-control (NTC) were also run in triplicate. The fold difference for each sample was obtained using the following equation: 2–ddCt. Ct is the threshold cycle.Total RNA was extracted from cells 48 h after transfection using Rezol  according to the manufacturer’s instructions. Subsequently, 4 µg RNA was converted to cDNA using SuperScript III, oligo (dT) primers, and PCR using the following primers: HBx Forward The lines of HBx transgenic mice used in this study were established and described elsewhere Institutional Review Board of the Human Investigation Committee of College of Medicine, National Cheng Kung University approved the study. Written informed consent was obtained from patients participating in this study. A total of ninety-five patients admitted to National Cheng-Kung University Hospital  with HBV-associated HCC who received curative surgery between 1 January 2003 and 31 December 2006 were enrolled, and samples of their resected liver tumor tissue were assembled in a tissue microarray. All 95 patients showed positive for serum HBV surface antigen and for HBx in tumor specimens analyzed using PCR, and negative for antibodies to the hepatitis C virus. The patients were regularly followed up at clinic visit every 1 to 3 months after curative surgery. The patients included 70 (73.7%) males and 25 (26.3%) females with age range of 44 to 77 years (mean age 60.7 years). The median follow-up time was 38 months . At the end of the follow-up, 28 patients had died of disease. The 1-year disease specific survival rate was 92.1% and 3-year disease specific survival rate was 74.2%.2 test, Fisher’s exact test, or Kaplan-Meier survival test. Significance was set at P<0.05.Immunohistochemical staining for pIKKβ, pTSC1, and pS6K1 protein expression was examined on adjacent 4-µm formalin-fixed paraffin-embedded tissue sections. An experienced gastrointestinal pathologist reviewed all specimens. Amino-ethylcarbazole chromogen was used for visualization. The positive protein staining was only considered in cytoplasmic immunoreactivity of cancer cells on a semi-quantitative scale that combined staining intensity and percentage of positively stained cells. Staining intensity was evaluated as low (0–10% positive cancer cells) or high (>10% positive cancer cells). Statistical analyses were done using a χWe established human hepatoma Hep 3Bx and Hep G2x cell lines which stably express HBx to investigate the effects of HBx overexpression on IKKβ/TSC1/mTOR signaling. The expression of HBx in Hep3Bx and Hep G2x was confirmed by both RT-PCR and Western blotting analysis . Next, wWe found that HBx enhanced basal and TNF-α-stimulated IKKβ and mTOR activity, and concomitantly increased phosphorylation and inactivation of TSC1 . We hypoSince activation of mTOR pathway could up-regulate the angiogenesis process To further understand the contribution of the HBx-modulated IKKβ/TSC1/mTOR signaling pathway in liver tumorigenesis, we used an HBx transgenic mouse model which has been shown to develop liver tumors To validate the relevance of the upregulation of TSC1/mTOR pathway via IKKβ signaling in HBV-associated human HCC, we evaluated the expression of pIKKβ, pTSC1, and pS6K1 in a tissue microarray of 95 HBV-associated human HCC tissue specimens by immunohistochemical staining. All 95 patients who received curative surgery were positive for both serum HBV surface antigen and HBx, but negative for antibodies to hepatitis C virus (data not shown). Expression of pIKKβ, pTSC1, and pS6K1 was detected in human HCC tissue specimens . AnalysiThe link between HBV and the development of hepatocellular carcinoma has been well established, but the pathogenic mechanism responsible for the transformation of normal hepatocytes to HCC is still far from understood, especially in view of the contribution of HBx to cancer progression. We have provided evidence that HBx activates IKKβ, which leads to inactivation of TSC1 and activation of mTOR/S6K1 and to the production of angiogenesis factor VEGF-A in HBx expressing hepatoma cells and liver tissues of HBx transgenic mice. The HBx-associated IKKβ/TSC1/mTOR signaling pathway may play a molecular switch that allows HBV-related HCC tumor progression. This is likely to be clinically relevant to pathogenesis as we found a statistically significant correlation between the phosphorylation of IKKβ and phosphorylation of TSC1, and between the phosphorylation of IKKβ and phosphorylation of S6K1 in HBV-associated HCC specimens.Chronic hepatitis B virus (HBV) infection causes an inflammation process in the normal liver tissue, resulting in liver damage that may subsequently evolve into liver cirrhosis and tumor development. The proinflammatory cytokine TNF-α has been shown to play a promoting role for tumor development We demonstrated that treatment of liver cancer cells with mTOR inhibitor rapamycin and IKKβ inhibitor Bay11-7082 effectively blocked the HBx-induced cell proliferation and VEGF-A production. Currently available treatments for non-operable advanced HCC patients, including local ablation therapy (radiofrequency ablation and percutaneous ethanol injection), trans-arterial chemo-embolization (TACE), chemotherapy and radiotherapy, are ineffective, resulting in very poor survival rates of patients In conclusion, we demonstrate that HBx deregulates TSC1/mTOR signaling through IKKβ and renders liver cancer cells more sensitive to TNF-α stimulation in activating mTOR downstream S6K1 activity through IKKβ signaling. Activation of the IKKβ/mTOR pathway occurs concomitantly with increased cell proliferation and angiogenesis, which may associate with the progression of the HCC. Consistently, blocking IKKβ or mTOR signaling with Bay 11-7082 or rapamycin, respectively, inhibits the liver cancer cell growth and VEGF-A production, suggesting that inhibitors of IKKβ or mTOR signaling may be useful as new therapeutics for the treatment of HBV-associated HCC.


Wnt/β-catenin pathway, the effectors of which are transcription factors of the LEF1/TCF family, is primarily associated with development. Strikingly, however, some of the genes of the pathway are schizophrenia susceptibility genes, and the proteins that are often mutated in neurodegenerative diseases have the ability to regulate β-catenin levels. If impairment of this pathway indeed leads to these pathologies, then it likely plays a physiological role in the adult brain. This review provides an overview of the current knowledge on this subject. The involvement of β-catenin and LEF1/TCF factors in adult neurogenesis, synaptic plasticity, and the function of thalamic neurons are discussed. The data are still very preliminary and often based on circumstantial or indirect evidence. Further research might help to understand the etiology of the aforementioned pathologies. T. T41]. TN-terminus after stimulation. Only a C-terminus-specific antibody could detect β-catenin in the nuclei of stimulated hippocampal neurons, implying that exclisively the truncated pool translocates [2+-activated protease. Application of the Calpain inhibitor MDL28170 abolished not only cleavage but also translocation in a Wnt-independent manner, indicating a new mechanism of β-catenin regulation in stimulated neurons. The authors proposed that the cleavage led to the accumulation of β-catenin by preventing its degradation because the truncated isoform could not be phosphorylated by GSK3α/β and consequently could not be targeted to the proteasome  were found to be upregulated after tetanic stimulation or glutamate treatment [An expected effect of the nuclear translocation of β-catenin in excited neurons is the activation of plasticity-related genes. A set of known β-catenin/TCF targets , Gabra2 (GABA receptor gene), and Calb2  in the thalamus [Cacna1g promoter in particular was shown to be activated by LEF1 and β-catenin in a luciferase assay and to bind LEF1 in a footprinting test [Cacna1g encodes the Cav3.1 subunit of so-called T-type channels that are critical for the function of thalamocortical relay neurons. T-type channels underlie the principal electrical property of these neurons, i.e., the intrinsic possibility to fire in two modes [Gene profiling in the forebrain revealed that voltage-gated ion channel and neurotransmitter receptor genes, which have at least two LEF1/TCF motifs in their putative regulatory sequences, are preferentially expressed in the thalamus . A loss-thalamus . The Cacing test , confirmwo modes . This fewo modes . Importawo modes . The conKnowledge about the role of canonical Wnt signaling effectors in the adult brain is still relatively superficial and inconclusive. Many studies have focused on the activation or inhibition of the canonical Wnt pathway while assuming concomitant changes in nuclear β-catenin activity and the availability of LEF1/TCF factors. In turn, when directly targeting β-catenin, research faces a major difficulty when attempting to delineate its adhesive and nuclear functions in a given cellular or physiological process. Wnt research suffers from insufficient tools to inhibit the nuclear canonical Wnt pathway and a lack of sophisticated animal models. The hope is that such tools and models will be soon be developed and implemented in fundamental and application neuroscience.


Endoscopy in IBD has tremendous importance to diagnose inflammatory activity, to evaluate therapeutic success and for the surveillance of colitis associated cancer. Thus it becomes obvious that there is a need for new and more advanced endoscopic imaging techniques for better characterization of mucosal inflammation and early neoplasia detection in IBD. This paper describes the concept of advanced endoscopic imaging for the diagnosis and characterization of Crohn's disease, including magnification endoscopy, chromoendoscopy, balloon-assisted enteroscopy, capsule endoscopy, confocal laser endomicroscopy, and endocytoscopy.  It is now well accepted that both patient groups are at an increased risk of developing colitis-associated cancer (CAC) . Chronics in IBD . While all patients with IBD suffer from an increased risk for developing CAC, it was shown that the duration and anatomical extent of disease are well-established risk factors for cancer development. Correspondingly, patients with a course of disease longer than 10 years or pancolitis are at an increased risk . TherefoNevertheless, to date, no randomized controlled study could show a reduced risk of CAC development by surveillance colonoscopy in IBD . In thisThus it becomes obvious that there is a need for new and more advanced endoscopic imaging techniques for surveillance in IBD. In recent years, new emerging endoscopic imaging techniques were introduced, allowing a detailed analysis of more mucosal and submucosal details. This paper describes the concept of advanced endoscopic imaging for the diagnosis and characterization of Crohn's disease.Magnification endoscopy utilizes a movable lens to vary the degree of magnification up to 150-fold, thereby allowing to characterize the mucosal surface  [Chromoendoscopy is divided into dye-based chromoendoscopy (DBC) and dye-less chromoendoscopy (DLC). DLC is further comprised of optical chromoendoscopy and virtual chromoendoscopy. The basic principle of chromoendoscopy is to enhance the mucosal detail and/or submucosal vascular network with the use of various dyes or endoscopic/optical and computer-based color programs. The contrast enhancement of the mucosal surface often results in an improved detection of subtle lesions. DBC uses different dye agents which are divided into absorptive agents , contrast agents  and reactive staining agents . Dye agents are mostly applied via standard spraying or plain biliary ERCP catheters . VariousTypical endoscopic aspects of conventional endoscopy and chromoendoscopy were recently summarized by the group from the Hyogo College of Medicine in Japan .in vivo. The same group prospectively analyzed indigo carmine-assisted high-magnification chromoscopic colonoscopy for the detection and characterisation of intraepithelial neoplasia in UC. Significantly more intraepithelial neoplastic lesions were detected in the magnification chromoendoscopy group compared with controls. Moreover, chromoendoscopy increased the number of flat lesions with intraepithelial neoplasia detected compared with controls [Hurlstone and coworkers analyzed high-magnification chromoscopic colonoscopy in UC as a tool for reliable assessment of disease extent . 325 patcontrols . Anothercontrols . Multivacontrols . Pancolonic chromoendoscopy and targeted biopsies of suspicious lesions represent a more effective surveillance method in IBD than taking only multiple nontargeted biopsies. Magnification chromoendoscopy improves the detection of preneoplastic and neoplastic mucosal changes. Magnification endoscopy has the potential to predict relapse in patients with quiescent disease.DLC is divided into optical chromoendoscopy including narrow band imaging  was introduced in 2002. Currently, various capsule systems from different companies are available for examination of the esophagus, small bowel, and colon . The capIn case of suspected small bowel, crohn's disease without strictures or stenosis capsule endoscopy should be preferred. Balloon-assisted enteroscopy offers the potential to perform interventions within the small bowel and is associated with a low complication rate.in vivo histology during ongoing endoscopy  and one represents a stand-alone confocal probe which is capable of passing through the working channel of most standard endoscopes  [Confocal laser endomicroscopy (CLE) was introduced in 2004 and has rapidly emerged as a promising approach to obtain real time ndoscopy . The tec France) .κ = 0.91) [Kiesslich and coworkers impressively demonstrated that combination of chromoendoscopy  and endomicroscopy could detect 4.75-fold more neoplasias in surveillance colonoscopy of patients with UC compared to conventional endoscopy. Moreover, 50% less biopsy specimens were required and CLE could predict neoplastic changes with a sensitivity, specificity, and accuracy of about 95%, 98%, and 98%, respectively . Also CL = 0.91) . One study by Li and coworkers proposed a classification system of inflammatory activity in IBD based on a 4-grade classification system of crypt architecture, as well as by analysis of microvascular alterations and fluorescein leakage. Both, assessment of crypt architecture and fluorescein leakage with CLE showed good correlations with histological results. Moreover, CLE seemed to be more accurate than conventional white-light endoscopy for evaluating macroscopically normal appearing mucosa. More than half of the patients with normal mucosa seen on conventional white-light endoscopy showed acute histologic inflammation, whereas no patients with normal mucosa or with chronic inflammation seen on CLE showed acute inflammation on histology .in vivo diagnosis of Crohn's-disease-associated histological changes. Endomicroscopy was able to diagnose Crohn's-disease-associated changes with high accuracy. Furthermore, endomicroscopy could detect residual macroscopic nonvisible mucosal inflammation as precisely as histology  [Our group evaluated endomicroscopy for ues 0.8) .CLE can detect more neoplasms in surveillance colonoscopy of patients with IBD and could predict neoplastic changes with high accuracy. CLE can reliably predict inflammatory activity in IBD during ongoing endoscopy, even in patients with macroscopically uneventful mucosa.in vivo microscopic imaging at a magnification up to 1390-fold magnification, thereby allowing the analysis of mucosal structures at the cellular level. EC is based on the principle of contact light microscopy and therefore only allows visualisation of the very superficial mucosal layer [Endocytoscopy EC  enables al layer . The tecal layer , 48. In al layer , 50. Oural layer .EC harbours the potential to accurately determine various inflammatory mucosal cells during ongoing endoscopy in IBD and thus the severity of the inflammation. where no one has gone before. Moreover, recent advances in endoscopic imaging now enable the endoscopist to obtain real time in vivo histology during ongoing endoscopy by using endomicroscopy or endocytoscopy. These emerging imaging modalities enable the endoscopist to detect and characterize more preneoplastic and neoplastic lesions and to predict mucosal inflammation more precisely as compared to conventional white light endoscopy. Recent efforts have been made to introduce molecular imaging in IBD by highlighting inflammatory cells and specific receptors. Results of these trials are highly anticipated and might open new avenues for diagnostic and therapeutic strategies in IBD.Advanced endoscopic imaging in IBD has seen a major innovation revolution during the last 10 years, including both vital and virtual chromoendoscopy. By using capsule endoscopy or balloon-assisted enteroscopy, the endoscopist is now able to evaluate the entire small bowel and to perform endoscopic therapies at a place


Loss-of-function experiments using morpholinos against PRMT4/CARM1 and PRMT5 combined with in situ hybridization, quantitative polymerase chain reaction, as well as immunohistochemistry indicate a positive, but differential, role of these enzymes during myogenesis in vivo. While PRMT5 regulates myod, myf5 and myogenin expression and thereby slow and fast fiber formation, PRMT4/CARM1 regulates myogenin expression, fast fiber formation and does not affect slow fiber formation. However, our results show that PRMT4/CARM1 is required for proper slow myosin heavy chain localization. Altogether, our results reveal a combinatorial role of PRMT4/CARM1 and PRMT5 for proper myogenesis in zebrafish.In vertebrates, skeletal myogenesis involves the sequential activation of myogenic factors to lead ultimately to the differentiation into slow and fast muscle fibers. How transcriptional co-regulators such as arginine methyltransferases PRMT4/CARM1 and PRMT5 control myogenesis  However, the mechanisms by which these enzymes control myogenesis have not been addressed yet. Indeed, no other report has addressed the precise role(s) of CARM1 and PRMT5 in vivo during development and particularly in myogenesis. To gain insights into their function in vivo, we turned to zebrafish to analyze their shared and divergent roles in myogenesis.The proper control of gene expression is essential for normal growth, development and differentiation. An early step in the process of gene activation consists in modifying histones and unraveling DNA sequences within promoter regions thereby facilitating the recruitment of specific and general transcription factors. Skeletal muscle differentiation involves cooperation between the muscle regulatory factors (MRFs) Myod, Myf5, Mrf4 and Myogenin myf5 and myod, but not myogenin, are required and cooperate to drive myoblast progenitor  commitment to the slow fiber type myf5 and myod but also of myogeninmyogenin expression whereas PRMT5, controls the expression of the early genes myod and myf5. Accordingly, we demonstrated that these enzymes affect proliferation negatively and act in myogenic differentiation. We subsequently showed that both PRMT5 and CARM1 control fast muscle fiber formation whereas PRMT5 triggers slow muscle fiber formation. Finally, we found that if a decrease of CARM1 expression was not affecting the initial step of slow muscle fiber formation it lead to a defect in their localization. Hence, our results indicate that PRMT5 and CARM1 play a combinatorial role in zebrafish myogenesis.In zebrafish, cells are committed to the myogenic fate through the activation and the cooperation of MRFs and Mef2 proteins CARM1 and PRMT5 during zebrafish development was first analyzed by in situ hybridization. We found that both CARM1 and PRMT5 were robustly expressed at 2-cell stage indicating that they are maternal genes . PRMT5 expression was not detected in the adaxial progenitor cells and was enriched in the medial part of the somite, whereas CARM1 was ubiquitously expressed in the somite . These phenotypes were rescued at both stages when CARM1 or PRMT5 mRNAs were co-injected with their cognate morpholino . Expression of endogenous CARM1 and PRMT5 was strongly reduced after injection of their respective morpholino, as confirmed by western blot . Phenotyrpholino . AltogetCARM1 and PRMT5 were expressed in the shield during gastrulation (goosecoid/gsc (data not shown), or of the mesodermal gene spadetail/spt/tbx16 , a slow fiber specific gene and of fast myosin light chain (mlc2f), a fast fiber specific gene in PRMT5- and CARM1-depleted embryos as compared to control embryos. In PRMT5 morphants, mlc2f was dramatically reduced as compared to control morphants  as well as myogenin, whereas CARM1 is required for fast fiber formation by regulating myogenin expression. Interestingly, our data also show that if CARM1 has no impact on slow fiber formation it is required for their proper localization.In summary, this study demonstrates that CARM1 and PRMT5, two transcriptional co-regulators that have been shown respectively to act positively and negatively on proliferation in cell-based assays, are both inhibiting proliferation and are required for myogenic differentiation in a whole organism. However, they play distinct roles in myogenesis: PRMT5 regulates slow and fast fiber formation by controlling early myogenic genes , into pBluescript KS  using EcoRI/HindII (zCARM1) and XmaI/HindIII (zPRMT5) and were transcribed with T3 polymerase. For cross-sections, embryos were embedded in 3% Low Melting Agar after whole-mount in situ hybridization and 30 µm sections were performed using a Leica vibratome (VT 1000S). For immunohistochemistry, the following antibodies were used: F310 fast Myosin Light Chain (DSHB), F59 slow Myosin Heavy Chain (DSHB) and phospho-Histone H3 (ser10)  with appropriate Alexa Fluor-conjugated secondary antibodies . Nuclei were stained with TO-PRO3  according to the manufacturer's protocol. Embryos were dissected, flat-mounted in glycerol or mounted in Mowiol and images were recorded on a microscope (NIKON Eclipse 80i) using a 20× Plan Apo na 0.5 or a 40× plan Apo na 1 with the NIS-element AR 2.30 software; or on a confocal microscope  with a 20× Plan Apo na 0.7 objective (zoom ×4) using the scanner resonant mode. Confocal images are stacks of the anterior somites of 14 or 18 ss embryos as stated in figure legends. For whole embryos, imaging was performed using a stereomicroscope (Leica MZ FL III) with the ACT-1C software.Whole-mount Total RNAs were extracted from 25 embryos at 14 ss or 18 ss as stated in figure legends with the RNeasy mini kit , eluted in RNAse free water and reverse-transcribed with the Superscript II Reverse Transcriptase  according to the supplier's instructions.EF1 mRNA copies. Primer sequences were the following: myod fw 5′- GCCCAAAGTGGAGATTCTGA-3′, rev 5′- GCCCATAAAATCCATCATGC-3′; myf5 fw 5′-GAGAGCATGGTTGACTGCAA-3′, rev 5′-GAATCACTTCCGGTTGGAGA-3′; myogenin fw 5′-AAACCATCTCCATCGTCCAG-3′; rev 5′-GGGTTCATCAATGT GCTCCT-3′; Mef2c fw 5′-GGTCTCTCCAGGGAACATGA-3′, rev 5′-GCCCATCACTT CTCCAGGTA-3′; EF1-alpha fw 5′-GATGCACCACGAGTCTCTGA-3′, rev 5′-TGATGACCT GAGCGTTGAAG-3′.Q-PCR analyzes were performed on an i-Cycler device  with the SYBR green JumpStart Taq Ready mix , according to the manufacturer's instructions. All experiments included a standard curve. Samples were analyzed in triplicates and the mean and standard deviation were calculated. Samples were normalized to the number of Whole cell extracts from 25 zebrafish embryos were classically prepared in 50 µl Laemmli sample buffer. 10 µl of each sample (5 embryos) were loaded per lane and subjected to SDS-PAGE. The following commercial antibodies were used: anti-CARM1 , anti-PRMT5 , anti alpha-Tubulin , anti-Rabbit IgG HRP conjugate  and anti-Mouse IgG HRP conjugate .Figure S1PRMT5 and CARM1 regulate myogenic factor expression. (A–B) In situ hybridization of embryos with the indicated mRNA probe and injected with the indicated Mo (A) at the shield stage or (B) at 14 ss. (A) Animal view . (B) Dorsal flat-mounted embryos stained for myod with a magnified region to the right.(TIF)Click here for additional data file.Figure S2PRMT5 and CARM1 regulate specifically myogenic factors expression at 14-somite stage (14 ss). Whole-mount in situ hybridization of embryos injected at one-cell stage with the indicated morpholino (Mo). Experimentrs were done twice with n = 20 for each condition. Embryos were collected at 14 ss and were analyzed for myogenic factors expression by (A) in situ hybridization, lateral view, anterior to the left or by (B) real time PCR with standard deviations relative to a control morpholino. Q-PCR procedures are detailed in the methods section. (A) Asterisk, anterior expression of myf5 in CARM1 morphant; arrow, down regulation of myogenin expression in the posterior somites in PRMT5 morphant. (B) Error bars represent standard deviations. *, P<0.01; **, P<0.001; ***, P<0.0001; ns, not statistically significant.(TIF)Click here for additional data file.Figure S3Knock down of CARM1 or PRMT5 does not affect their mutual expression. One-cell stage embryos injected with either a control morpholino (MoCt), or a morpholino against PRMT5 (MoP5) or against CARM1 (MoC1) (left panels). Mos were co-injected with either PRMT5 mRNA (MoP5+P5) or CARM1 (MoC1+C1) (right panels). Embryos were collected at 14-somite stage and analyzed for CARM1 and PRMT5 expression by in situ hybridization. Note that PRMT5 expression is strongly enhanced in MoP5-injected embryos (white asterisk), which can be rescued by the co-expression of PRMT5 mRNA (black asterisk).(TIF)Click here for additional data file.Figure S4PRMT5 and CARM1 mRNAs rescue specifically myogenin expression affected by their cognate morpholino(*). One-cell stage embryos were injected with either a morpholino control (MoCt), or a morpholino against PRMT5 (MoP5) or CARM1 (MoC1), alone or in combination with either PRMT5 or CARM1 mRNA. Embryos were collected at 14-somite stage and were analyzed for myogenin expression by in situ hybridization. Experiments were done twice (n = 22 for each condition).(TIF)Click here for additional data file.Figure S5CARM1 (C1) and PRMT5 (P5) control myogenesis differentially.  One-cell stage embryos were injected with the indicated morpholino (Mo) or a control Mo (MoCt) and were analyzed by whole-mount immunohistochemistry for (A) slow fibers and (B) fast fibers at 18-somite stage. Lateral views, anterior to the left. Both slow and fast fiber formation require PRMT5 (n = 12). CARM1 is necessary for fast fiber specification but does not affect slow fiber specification (n = 15). Antibodies used were: F310 fast Myosin Light Chain (DSHB), F59 slow Myosin Heavy Chain (DSHB) and appropriate Alexa Fluor-conjugated secondary antibodies . Nuclei were stained with TO-PRO3  according to the manufacturer's protocol.(TIF)Click here for additional data file.