The level of AB073614 was found to correlate inversely with sex-determining region Y-box 7 (SOX7) expression but correlate positively with Wnt/β-catenin signaling activity. Of note, the data showed that the inhibition of SOX7 could reverse the tumor-suppressive effect of the silencing AB073614 on glioma in vitro and in vivo. Furthermore, the results indicated that AB073614 induced Wnt/β-catenin signaling activity by repressing SOX7 expression. In conclusion, the findings demonstrated that AB073614 promoted the progression of glioma by targeting SOX7 to activate the Wnt/β-catenin signaling pathway, suggesting that the inhibition of AB073614 might be a potential target for therapeutic intervention in glioma patients.Long noncoding RNA (lncRNA) AB073614 has recently shown to be aberrantly increased and identified as a poor prognostic biomarker in human glioma. However, the potential mechanisms remain unknown. This study demonstrated that AB073614 expression was significantly upregulated in both glioma tissues and cell lines, and glioma patients with high AB073614 expression had lower overall survival rates. Importantly, silencing AB073614 expression remarkably inhibited U251 cell proliferation, migration, and invasion  Glioma is the most common and aggressive form of adult primary brain tumors, accounting for 80% of malignant brain tumors , 2. AccoLong noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides in length with no protein-coding ability . DysreguThe Wnt/β-catenin signaling pathway is a highly conserved molecular mechanism that regulates a variety of biological processes, such as cell proliferation, differentiation, and morphogenesis in both normal development and disease progression –13. Morein vitro. However, the inhibition of SOX7 could reverse the tumor-suppressive effect of silencing AB073614. These results suggested that AB073614 promoted the progression of glioma by activating Wnt/β-catenin signaling through downregulating Sox7, and the inhibition of AB073614 might be a potential therapeutic approach for treating glioma.The present study aimed to investigate the role and possible molecular mechanism of AB073614 in human glioma evolution. The results showed that the expression of AB073614 was significantly increased in both glioma tissues and cell lines; it was inversely correlated with SOX7 but positively correlated with Wnt/β-catenin signaling activity. Importantly, silencing AB073614 could not only remarkably suppress Wnt/β-catenin transcription and its targeted genes, including Cyclin D1 and c-Myc, but also inhibit cell proliferation, migration, and invasion Here, we first investigated the expression level of AB073614 in human glioma tissues and cells. The expression level of AB073614 was detected in 89 paired human glioma and adjacent normal tissues, as well as in glioma cell line (U251) and normal human astrocytes (NHA), using quantitative real-time polymerase chain reaction (qRT-PCR). AB073614 expression was found to be significantly upregulated in glioma tissues compared with adjacent normal tissues, with a higher level in high-grade glioma Figure . In paraThe study then explored whether the Wnt/β-catenin signaling pathway was activated in human glioma. We isolated cytoplasm and nucleus, and measured the expression of β-catenin in nucleus, as well as two Wnt/β-catenin signaling targets Cyclin D1 and c-Myc in the whole cell by western blot assay in this study. The results showed that the level of nuclear β-catenin and its target gene (Cyclin D1 and c-Myc) expression was markedly increased in glioma tissues and cell lines compared with normal tissues or NHA, respectively Figure  and 2B. AB073614 expression was knocked down in U251 cells by transducing sh-AB073614 to examine the role of AB073614 in glioma progression. The MTT assay indicated that the decreased expression of AB073614 dramatically inhibited cell proliferation compared with the NC group Figure . Then, wEmerging studies have suggested that SOX7 is downregulated in several human cancers –21. TherAB073614 positively regulated the activity of Wnt/β-catenin signaling pathway and negatively regulated the SOX7 expression level in glioma cells. Given that SOX7 has been showed to remarkably downregulate Wnt/β-catenin transcription and inhibit the expression of Wnt target genes, it was hypothesized that AB073614 regulated Wnt/β-catenin signaling activity through SOX7. The relationship between SOX7 and Wnt/β-catenin signaling activity was first confirmed to test this hypothesis. U251 cells were transduced with NC, shRNA targeting SOX7 (sh-SOX7), or SOX7 overexpression (SOX7) lentiviral vector. The transduction efficiency was shown in Figure in vivo. U251 cells transduced with NC, sh-AB073614, or sh-AB073614 + sh-SOX7 lentiviral vector were inoculated subcutaneously into the flanks of nude mice. Tumor formation was then monitored for 28 days. It was found that the average size and weight of tumor in the sh-AB073614 group were much smaller than those in the NC group, indicating a significant tumor-suppressive effect of silencing AB073614 in vivo. In contrast, the tumor size and weight in the sh-AB073614 + sh-SOX7 group showed no difference compared with the NC group  were acquired from Beijing Zhongyuan Company  and grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% heat-inactivated fetal bovine serum , streptomycin (100 µg/mL), and penicillin (100 units/mL). All cell lines were maintained at 37°C in a humidified atmosphere containing 5% COProteins from glioma tissue samples and cell line (U251) were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred on to polyvinylidene membranes. The blots were incubated with Cyclin D1 primary antibody , c-Myc primary antibody , and β-catenin . Then, the blots were incubated with an appropriate second antibody that conjugated with the horseradish peroxidase for 2 h. Finally, the complexes were detected using chemiluminescence.Target genes were analyzed using an Mx4000 or Mx3005 multiplex qRT-PCR system. Then, the expression of AB073614 was normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level. The primary sequences used were as follows: AB073614: forward, 5ʹ-TCTGCTCCTGGGTCTTACAC-3ʹ; AB073614: reverse, 5ʹ-TGCAACCACATGTAACCACA-3ʹ; GAPDH: forward, 5ʹ-CCCATCACCATCT¬TCCAGGAG-3ʹ; GAPDH: reverse, 5’-GTTGTCATGGATGAC¬CTTGGC-3ʹ.U251 cells were seeded in 96-well plates. The cell viability was analyzed using MTT assay. After incubation, the cells were treated with MTT (5 mg/mL) for 4 h at 37°C. Then, the medium was removed and dimethyl sulfoxide was added to each well. The optical density values of the cells were determined on a microplate reader at 490 nm.Yang et al reported the methods of transwell assay . In brieThe cells were cultured in six-well plates and then incubated until 80% confluence before of wounding. Then, a 200-μL tip was used to make a vertical wound and the cells were washed three times with phosphate-buffered saline to remove the cell debris. Cell migration to the wounded region was observed by Nikon microscopy at the designated time point.P values < 0.05 were considered statistically different .All the statistical analyses were conducted using Statistical Package for the Social Sciences Version 20.0 software . One-way analysis of variance (ANOVA) followed by the Bonferroni test was used to analyze the data. The values were expressed as the mean ± standard error of mean. Survival analysis was carried out using the log-rank test in GraphPad Prism 5.


Musa acuminata. Its complex genome decoded by hybrid sequencing strategies revealed panoply of genes and transcription factors involved in the process of sucrose conversion that imparts sweetness to its fruit. Historically, banana has faced the wrath of pandemic bacterial, fungal, and viral diseases and multitude of abiotic stresses that has ruined the livelihood of small/marginal farmers’ and destroyed commercial plantations. Decoding structural genome of this climacteric fruit has given impetus to a deeper understanding of the repertoire of genes involved in disease resistance, understanding the mechanism of dwarfing to develop an ideal plant type, unraveling the process of parthenocarpy, and fruit ripening for better fruit quality. Further, injunction of comparative genomics will usher in integration of information from its decoded genome and other monocots into field applications in banana related but not limited to yield enhancement, food security, livelihood assurance, and energy sustainability. In this mini review, we discuss pre- and post-genomic discoveries and highlight accomplishments in structural genomics, genetic engineering and forward genetic accomplishments with an aim to target genes and transcription factors for translational research in banana.Evolutionary frozen, genetically sterile and globally iconic fruit “Banana” remained untouched by the green revolution and, as of today, researchers face intrinsic impediments for its varietal improvement. Recently, this wonder crop entered the genomics era with decoding of structural genome of double haploid Pahang (AA genome constitution) genotype of  It rank 1a,b,c) . This fr 1a,b,c)  in terms 1a,b,c) .Musa  that comprises “Eumusa” with 22 chromosomes and “Australimusa” with 20 chromosome sets . As per the International Classification System (ICS) Musa has five major sections, viz., Eumusa, Rhodochlamys, Callimusa, Australimusa and Incertae sedis. Of these sections, Eumusa contributes to the wide diversity for wild and cultivated types. There are 11 wild species reported across the globe of which six species are prevalent in India. M. acuminata and M. balbisiana are the major progenitors of present day bananas and have a wider distribution throughout the banana growing states including Andaman and Nicobar Islands.Banana is considered to have originated in Asiatic region  with SouMusa spp. section Eumusa) originated from intra and inter-specific hybridization between two wild species namely M. acuminata Colla (A genome) and M. balbisiana Colla (B genome). Polyploidy and hybridization have given rise to a number of diploid, triploid, and tetraploid clones with different combinations of A and B genomes like AA, AB, AAA, AAB, ABB, AABB, AAAB, ABBB  cultivars. It is believed that chromosome restitution at meiosis in M. acuminata gave rise to edible triploid AAA (acuminata) bananas. Though not clear, using variation in anthocyanin pigments as a marker, it is assumed that three different subspecies such as malaccensis, banksii, and zebrina  and B  genomes. The triploid genetic constitution of banana posed a huge challenge to the sequencing and assembly of its genome. Therefore, a double haploid (PH-Pahang) M. acuminata was generated and sequencing was accomplished in 2012 by a team of researchers (Musa ranges from 554 megabase (Mb) in M. balbisiana to 523 Mb in M. acuminata. The genome is two/three times larger than the genomes of cultivated rice  or Arabidopsis (150 Mb) but still is much smaller than the Triticeae cereals such as wheat , barley  and rye . Further, 70% of the assembly anchored to 11 Pahang chromosomes with 250 scaffolds covering 92% of the annotated genes. Sequencing information further revealed that banana genome contained approximately 36,000 genes which were mostly located in the distal-end of the chromosomes. It was also observed that like most plant genomes (Musa genome consists of repetitive DNA (about 50%) comprising of transposable elements.Genetically, banana is a triploid crop and comprises of both A  is highest among all sequenced plants. Of these, 759 TFs were specific to banana and belonged to the MYB and AP2/ERF transcription factor families that plays a role in fruit architecture and ripening  identified in ripening . ExpressFigure 1e) caused by the fungus Fusarium oxysporum sp. cubense has inflicted major devastation to the banana plantations , caused by Mycosphaerella fijiensis which results in more than 50% crop losses  grape stilbene synthase (StSy), and (iii) superoxide dismutase Cu, Zn-SOD from tomato were used to transform Grand Nain banana . The famous high-yielding semi-dwarf variety of rice (IR8) which ushered in the ‘Green Revolution’ was accomplished due to a mutation in the eveloped  that exheveloped  to tinkeP or P1) interacting with minor ones (Banana garners huge demand in human consumption due to its seedless fleshy fruit. The ability of plants to bear seed-less fruit (without pollination and fertilization) is due to the phenomenon known as “Parthenocarpy.” Additionally, female sterility is also hypothesized to be the cause of parthenocarpy in many fruits including banana , 1962. Pnor ones . With deM. balbisiana) has been associated with banana pre-harvest development and post-harvest ripening  when fruits taste/smell tarter. Being a climacteric fruit, ripening in banana is an important phenological event. Edible cultivated bananas are triploids with AAA, AAB and ABB genomic composition of which the A-genome  resistance to infection of Xanthomonas and (ii) tolerance to drought/water stress (Brachypodium distachyon (Due to inherent rapid rate of growth, shallow root system and permanent green canopy, banana requires abundant supply of water  for optir stress . At moler stress , beans (r stress , brachypr stress  and ricer stress . In factr stress . Gene exr stress , Brachypstachyon  and maizstachyon . Post-gestachyon , microRNstachyon  along wistachyon  is expecInter alia, being the most consumed fruit banana has immense capability to contribute to food security through higher and stable yields. It has been a challenge for banana breeders to produce disease resistant polyploid hybrids through genetic recombination. Like other crops (Musa researchers to apply functional genomics and comparative genome analysis to identify, clone, and characterize genes responsible for key agronomic traits as well as biotic and abiotic stress tolerance. As of today, the mechanism of action and the inter-connection of various signaling cascades remain largely unknown in banana. Functional analysis to unravel regulatory details small-, micro-, and si-RNAs and their role in the stress responses and hormone signaling will provide useful information for translational research in banana. As our molecular understanding advances, biotechnological interventions such as gene discovery, genetic engineering, targeted transformation/regeneration, and progressive bioinformatic tools (Banana has remained practically untouched by the ‘Green Revolution’ because of the difficulties encountered in conventional breeding programs in this genetically sterile triploid crop. er crops , availabic tools  coupled ic tools  will accPD planned, collected information, organized, and prepared the manuscript. RR designed and contributed for writing and reviewing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Among nine SNVs, we explored the functional impact of the de novo mutation in TBL1XR1 [c.30 C > G (p.Phe10Leu)], a gene previously found to be associated with autism spectrum disorder and epilepsy. Protein structural analysis revealed that Phe10Leu mutation may decrease the structural stability of the TBL1XR1 protein. We demonstrate that Phe10Leu mutation alters the interaction of TBL1XR1 with N-CoR and β-catenin, which play critical roles in regulation of Wnt-mediated transcriptional activity. Consistently, TBL1XR1-mediated activation of Wnt signaling was up-regulated by Phe10Leu mutation. These results suggest that a de novo TBL1XR1 point mutation could alter Wnt/β-catenin signaling activity. Further studies are required to clarify the involvement of TBL1XR1 mutations in neuropsychiatric conditions.Here we report  A meta-analysis of twin studies revealed that the point estimate of heritability of schizophrenia is 81%2. To date, multiple common and rare genetic variants have been identified as genetic risk factors for schizophrenia5, while there are a number of patients with no family history of schizophrenia, so called sporadic cases.Schizophrenia is a complex condition resulting from genetic and environmental etiological influences6. De novo mutations, such as single-nucleotide variants (SNVs), insertions and deletions (INDELs), and copy-number variants (CNVs) may contribute to the genetic etiology of sporadic schizophrenia and may explain the high prevalence rate of schizophrenia in general population. Recent trio-based studies using next-generation sequencing technology have identified de novo SNVs in sporadic schizophrenia13. Nonetheless, these rare SNVs are uncommon across studies, and molecular mechanisms of these mutations underlying schizophrenia still remain obscure. No whole-exome sequencing studies of sporadic schizophrenia have been previously reported in the Japanese population.Lynch (2010) estimated that the average newborn acquires a total of 50–100 new mutations, resulting in approximately 0.86 novel amino acid-altering mutations per generationde novo non-synonymous SNVs. We further examined the effect of the novel de novo TBL1XR1 mutation [c.30 C > G (p.Phe10Leu)] on the protein structure of TBL1XR1 and Wnt/β-catenin signaling pathway.In the present study, we conducted whole exome sequencing of 18 trios consisting of patients with sporadic schizophrenia and their parents, and we identified de novo mutations, we found 82 de novo SNVs in 18 trios. Of these 82 de novo SNVs, 17 were predicted to be non-synonymous mutations. Of these 17 de novo non-synonymous SNVs, we validated nine mutations in eight trios by Sanger sequencing , member 4] gene and one in the TBL1XR1 [transducin (beta)-like 1 X-linked receptor 1] gene, were predicted as damaged by all of three software tools  and West syndrome15, we conducted subsequent structural and functional analyses of the observed de novo non-synonymous TBL1XR1 mutation [c.30 C > G (p.Phe10Leu)]. The TBL1XR1 point mutation (p.Phe10Leu) was not observed either in the independent 1,191 patients with schizophrenia nor in the 1,986 non-psychiatric control subjects.We conducted exome sequencing of 18 trios. On average, we obtained 15.4 GB of raw sequence data per sample, and 96.9% of these data were mapped to the reference genome (hg19). On detection of TBL1XR1 point mutation (p.Phe10Leu) on the protein structure of TBL1XR1  and tryptophan-aspartic acid 40 (WD40) repeat. The Phe10Leu mutation is located within the NTD. To assess the substitution in the context of protein structure, we built structural models of the NTD of the control and Phe10Leu TBL1XR1, and we obtained tetramer models of the NTD  was exogenously expressed in 293T cells and co-immunoprecipitated with N-CoR and β-catenin. While the Phe10Leu mutation disturbed the interaction between TBL1XR1 and N-CoR, we observed increased binding of TBL1XR1 to β-catenin  and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) protein complex, which regulates transcriptional repression machinerynin Fig. . ConsistLeu Fig. . Next, wion Fig. . Interesion Fig. .Figure 213. While no genes reported in previous trio-based genetic studies of schizophrenia13 was detected in our cohort, we identified the de novo mutation in TBL1XR1 (p.Phe10Leu), a gene previously found to be associated with neuropsychiatric conditions. O’Roak et al. reported two de novo point mutations (p.Leu282Pro and p.Ile397SerfX19) in sporadic cases of ASD20. Saitsu and colleagues found a de novo point mutation (p.Gly70Asp) in a patient with West syndrome and three missense variants in this gene  in patients with epilepsy15. Deletions on 3q26.32, encompassing the TBL1XR1 gene, have been reproducibly associated with intellectual disability22. These results suggest that mutation of the TBL1XR1 gene may contribute to a genetic vulnerability to multiple neurodevelopmental psychiatric conditions.To the best of our knowledge, this is the first study conducting trio-based exome sequencing using Japanese subjects with sporadic schizophrenia. The observed exome point mutation rate in schizophrenia in the present study was similar to those of previous exome sequencing studies25. Recent studies demonstrated that TBL1XR1 also plays a critical role in recruiting β-catenin to Wnt target gene promoters for transcriptional activation17. These results suggest that TBL1XR1 may act as a molecular switch for transcriptional activation and repression, which may be regulated by its posttranslational modification, such as SUMOylation26. The results of our protein structural analysis indicate that Phe10Leu substitution in the NTD of TBL1XR1 decreases structural stability of the NTD, which may influence binding to other components of the corepressor complex. In fact, we observed that Phe10Leu substitution decreases interaction between TBL1XR1 and N-CoR, whereas binding of TBL1XR1 with β-catenin is increased, which may explain observed up-regulation of Wnt/β-catenin-mediated transcriptional activity induced by Phe10Leu substitution.The results we obtained in protein structural and functional analysis suggest potential pathogenic impact of Phe10Leu mutation in TBL1XR1 function. TBL1XR1 is a component of the quaternary corepressor complex composed of N-CoR, SMRT and histone deacetylase 3 (HDAC3), which plays a key role in regulating transcription repression28. Up-regulation of Wnt/β-catenin signaling and dysregulated function of NCoR have been documented in multiple types of cancers30. High expression of TBL1XR1 has been reported to be associated with a poor prognosis of colorectal cancer31. Consistently, Wnt/β-catenin signaling and the N-CoR/TBL1XR1 complex play critical roles in cell proliferation and differentiation29. Thus, it would be of interest to investigate how altered Wnt signaling pathway induced by TBL1XR1 mutations may affect cellular processes in brain development.Accumulating evidences suggest that altered Wnt signaling may be implicated in etiopathophysiologies of neurodevelopmental psychiatric conditions, such as schizophrenia and ASDABCD4 point mutation (p.lle344Thr) found in the present study was also predicted as damaged by in silico functional analysis. ABCD4 is an ABC transporter that has been classified as a member of the D subfamily of peroxisomal ABC transporters, and mutations in ABCD4 cause a new inborn error of vitamin B12  metabolism32. Given that decreased levels of cobalamin have been reported in the frontal cortex in schizophrenia and autism compared with controls33, the effect of ABCD4 point mutations in the metabolic processes of cobalamin in brains should be investigated in future studies.The de novo non-synonymous SNVs as a result of the whole-exome sequencing of 18 trios. In particular, a de novo TBL1XR1 point mutation could alter Wnt/β-catenin signaling activity, supporting the potential involvement of altered Wnt signaling pathway in neurodevelopmental psychiatric disorders. Further studies are required to clarify the involvement of TBL1XR1 mutations in neuropsychiatric conditions.In conclusion, we report nine novel We recruited 18 trios of patients with schizophrenia and their unaffected parents from the Tokushima University Hospital, Ehime University Hospital, Kochi University Hospital, and Nagasaki University Hospital in Japan. Patients with schizophrenia  were independently recruited from Tokushima University Hospital in Japan. Schizophrenia was diagnosed according to Diagnostic and Statistical Manual of Mental Disorders (DSM)-IV criteria by at least two expert psychiatrists based on extensive clinical interviews and a review of medical records . Exome enrichment was performed by the TruSeq DNA Sample Prep Kits and TruSeq Exome Enrichment Kit . Exome sequencing was performed on a HiSeq1000/1500 .34. BWA-generated SAM files were converted into BAM format, sorted and indexed using SAMtools v.0.1.1935, then processed by Picard (v1.90) to mark duplicated reads. The BAM-formatted files were further processed using the Genome Analysis Toolkit  according to the GATK’s best-practice recommendations. In brief, the BAM files were processed with GATK tools  to perform local realignment around indels and recalibration of the base quality scores, followed by the data compression with the downstream GATK tool (ReduceReads). When using all of the processed BAM files from 18 trios, multi-sample variant calling with the UnifiedGenotyper tool in GATK (v2.6-5) was done to identify SNV and indel candidates. The resulting Variant Call Format file  was applied to variant quality score recalibration with the GATK VariantRecalibrator tool. Genomic annotations associated with the variants in the VCF file were added using snpEff (v2.0.5d) with the GRCh37.64 database. According to the GATK’s recommendations for variant filtering, the annotated VCF file was used to extract potential true positive variants on the following values: QD < 2.0, MQ < 40.0, FS > 60.0, HaplotypeScore > 13.0, MQRankSum < −12.5, and ReadPosRankSum < −8.0. After the multi-sample VCF file was split into each of the 18 trios using GATK, the trio-based VCF files generated were used to obtain variants that violated Mendel’s law of segregation in the respective families as candidate de novo variants. Of these variants, we extracted de novo SNV candidates on the following settings: 1) read depth of coverage at SNV sites were 30 or more in both the proband and parents from a trio of interest, 2) SNVs were not present in the dbSNP v137 database and 3) SNVs were seen in only the proband of interest. Candidate de novo non-synonymous SNVs were validated by standard Sanger sequencing on an ABI 3130xl DNA Analyzer. Genotyping of the TBL1XR1 point mutation [c.30 C > G (p.Phe10Leu)] was performed using a commercially available TaqMan probe with the Applied Biosystems 7500 Fast Real Time PCR System, according to the protocol recommended by the manufacturer .Raw sequencing data for each individual was mapped to the human reference genome (build hg19) by using the Burrows-Wheeler Aligner (BWA v0.5.9)de novo non-synonymous SNVs on protein function, PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/)36, SIFT (http://sift.jcvi.org/)37, and PROVEAN (http://provean.jcvi.org/index.php)38 were used.To predict the effect of 39. The 2xtc.pdb template was used for building the models. Models contained residues 2–75 of TBL1XR1. Folding energies of the models were calculated with FoldX40. Contact area for each residue was calculated by using PDBePISA41. Structural illustrations were depicted with Swiss PDB Viewer42.Structural models of the N-terminal domain (NTD) of TBL1XR1 were built with homology modeling method by using the Swiss Model17. The FLAG-tagged mutant TBL1XR1 (TBL1XR1Phe10Leu) expression construct was made by a PCR-based mutagenesis protocol43. For biochemical experiments, the following antibodies were used: rabbit polyclonal anti-β-catenin antibody , rabbit polyclonal anti-N-CoR antibody , as well as mouse monoclonal and rabbit polyclonal anti-FLAG antibodies .The FLAG-tagged TBL1XR1 expression construct was a gift from Dr. Cun-Yu Wang 6 293FT cells in a medium containing 1% FBS. Transfection of the TOPFlash reporter plasmid, Renilla luciferase cDNA in an SV40  and N-CoR expression construct, together with FLAG-tagged TBL1XR1, TBL1XR1Phe10Leu or an empty (mock) vector, was carried out with Lipofectamine 2000 . Two days after transfection, cells were lysed, and Wnt/β-catenin activity was measured using the Promega Dual Luciferase Reporter Assay System  and a FLUOstar Luminometer .Human embryonic kidney 293FT cells were maintained in Dulbecco’s modified Eagle’s medium nutrient mixture F-12 (DMEM/F12 (1:1); Gibco BRL, Gaithersburg, MD, USA) containing 10% FBS at 37 °C in a 5% CO2/95% air atmosphere. Twelve-well plates were seeded with 1 × 10Phe10Leu expression construct or mock vector were lysed in IP buffers  and , respectively. Supernatant fractions obtained after centrifugation at 12,000 g for 15 minutes were incubated with primary antibodies and protein G Plus/Protein A agarose . Immunoprecipitates were analyzed with SDS-PAGE followed by Western blotting after extensive washing. Endogenous β-catenin and N-CoR binding to exogenous TBL1XR1 and TBL1XR1 Phe10Leu was analyzed by densitometry.293FT cells and HT22 cells transfected with the FLAG-tagged TBL1XR1 or TBL1XR1Supplementary Figure1Supplementary Table S1


T850 and the regulatory PRMT5T80 residues as well as the symmetric dimethylation of H2A/4 were elevated in human hepatocellular carcinoma and in other types of cancers. These changes correlated positively with the grade and state of the tumors. Our results suggest the tumor suppressor role of MP via inhibition of PRMT5 thereby regulating gene expression through histone arginine dimethylation.Myosin phosphatase (MP) holoenzyme is a protein phosphatase-1 (PP1) type Ser/Thr specific enzyme that consists of a PP1 catalytic (PP1c) and a myosin phosphatase target subunit-1 (MYPT1). MYPT1 is an ubiquitously expressed isoform and it targets PP1c to its substrates. We identified the protein arginine methyltransferase 5 (PRMT5) enzyme of the methylosome complex as a MYPT1-binding protein uncovering the nuclear MYPT1-interactome of hepatocellular carcinoma cells. It is shown that PRMT5 is regulated by phosphorylation at Thr80 by RhoA-associated protein kinase and MP. Silencing of MYPT1 increased the level of the PRMT5-specific symmetric dimethylation on arginine residues of histone 2 A/4, a repressing gene expression mark, and it resulted in a global change in the expression of genes affecting cellular processes like growth, proliferation and cell death, also affecting the expression of the retinoblastoma protein and c-Myc. The phosphorylation of the MP inhibitory MYPT1 The molecular mechanism behind the pathogenesis of HCC is poorly understood, although molecular markers and more precise classification would be crucialNur77, a proapoptotic geneIt is assumed that free PP1c is not present in the cells, but it performs its diverse biological activities by forming holoenzymes with one of the almost 200 PP1c-binding proteins, termed targeting/inhibitory subunits, in a mutually exclusive mannerOne of the potential regulatory pathways related to MP function is driven by the RhoA GTPase and its downstream Ser/Thr kinase termed RhoA-activated kinase (ROK) which was shown to phosphorylate MYPT1 at Thr696 and Thr850, thereby causing inhibition of MP activityTo elucidate the contribution of the ROK/MP axis in tumorigenesis beyond their already described role in angiogenesis and metastasis our goal was to describe the nuclear function of MP. Our present data indicate a potential tumorsuppressor role for MP in human hepatocarcinoma cells via antagonizing the tumorigenic effect of ROK. Our results shed light on the involvement of ROK/MP enzyme pair in the phosphorylation/dephosphorylation of the protein arginine methyltransferase 5 (PRMT5) via regulating arginine methylation of histones thereby affecting signaling for cancer formation.G, NG-symmetric dimethylarginine (SDMA) as a type II PRMT. It has multiple cytosolic and nuclear binding partners and is implicated in the regulation of transcription, RNA transport and cellular signalingInitial reports have already described the presence of MYPT1 in the nucleus but its exact subnuclear localization and function remained unknown1-296 and anti-PRMT5 antibodies were used to immunoprecipitate target proteins and a possible association between MYPT1 and PRMT5 was assayed in HepG2 cells (667-1004). These MYPT1 species were bound on SPR sensorchips to immobilized anti-GST and purified FT-PRMT5 was flown through the flow cells as an analyte. PRMT5 formed a stable complex with the full-length GST-MYPT11-1004 in a concentration range of 0.3125–5 μM and the association constant for binding was Ka = (1.62 ± 0.29) × 107 M−1 (667-1004 (data not shown). Fitting the sensograms with a 1:1 binding model resulted in overlaps with the experimental curves suggesting a 1. 1 molar ratio in the GST-MYPT11-1004- PRMT5 complex formed. These data demonstrate also that PRMT5 forms a stable interaction with the N-terminal region of MYPT1.Since PRMT5 was identified as a nonspecific interacting protein in affinity purified Flag-tagged complexes from HeLa and tsa cell lysatesG2 cells . PRMT5 aG2 cells . Surface 107 M−1 , while nhttp://www.phosphonet.ca/). Protein kinase A (PKA), protein kinase C (PKC) and ROK were suggested as potential phosphorylating enzymes therefore they were probed in in vitro phosphorylation assays. The autoradiogram in 32P-ATP) was used as phosphoryl donor substrate. Western blot analysis of ROK-phosphorylated PRMT5 by antibody specific for phosphorylated Thr  was significantly decreased in the presence of H1152, a selective Rho-kinase inhibitor. Alanine mutant of PRMT5T80 (PRMT5T80A) was generated by site-directed mutagenesis and phosphorylation of this mutant was probed in ROK assay in the presence and absence of H1150. As judged with anti- anti-PPRMT5T80 antibody on Western blot no signal was detected confirming the Thr80 specificity of ROK in PRMT5 phosphorylation  on their common arginine 3, the so called “R3 motif”in vitro led us to examine whether MP plays a role in the regulation of gene expression through the arginine methylation of H2A and 4 at the gene repression sites in vivo. We first transfected HepG2 cells with MYPT1-specific siRNAs (siMYPT1) or non-targeting siRNA as a control. SiMYPT1 was able to lower the protein expression of MYPT1 by >70%  to a suppressive symmetric dimethylarginine (SDMA) mark at these motifs with respect to gene expression by >70%  without  by >70%  or the c by >70%  as signs by >70% . PRMT5 w by >70% . MYPT1-s by >70%  or on th by >70% . Moreove by >70%  and H4R3 by >70%  and it w by >70%  and H4R3 by >70%  increase by >70% . To veri by >70% . Knockinwww.figshare.com website. n = 3) in the siMYPT1 samples when compared to gene expression pattern. Hierarchical cluster analysis was performed on all differentially expressed genes using average linkage with Pearson’s dissimilarity and the number of induced and repressed genes were presented in a heat map  HepG2 cells . The comheat map . BioinfoThe evaluation of genes related to MYPT1-silencing revealed the significant downregulation of several tumor suppressors and transcription factors. One of them is the retinoblastoma protein (pRb) that plays a pivotal role in the negative control of the cell cycle and in tumor progression as a major regulator of G1 checkpointT80  and PRMT5 and it was verified that PRMT5 binds to the N-terminal region of MYPT1.In the present study we have characterized a novel mechanism of tumorigenesis governed by ROK and MP in hepatocarcinoma cells (HCC). We have identified Thr80 in PRMT5 as the target of ROK/MP and the phosphorylation/dephosphorylation of this site is shown to modulate the activity of PRMT5. At high phosphorylation of PRMT5 at Thr80 symmetric dimethylation of the histone R3-motif is preferred which accompanied by tumorogenesis, while dephosphorylation by MP has an opposite effect. Therefore, MP could be assumed as a tumorsuppressor in HCC. The interplay between MP and PRMT5 is also supported by interaction analysis. First, the presence of PRMT5 in the MYPT1 nuclear interactome is appealing for an interacting protein complex. Second, SPR-based in vitro  well conserved among vertebrates but not in invertebrate orthologs. Furthermore, PRMT5 exhibits no sequence homology with PRMT1-4 enzymesOur findings show that ROK/MP counteract on the Thr80 phosphorylation site of PRMT5 regulating its methyltransferase activity both in vitro  and in v applying the manufacturer’s protocol. FT-PRMT5wt and -PRMT5T80A proteins were bound to anti-Flag M2 affinity gel during kinase and phosphatase assays and in vitro protein arginine methyltransferase assays. FT-PRMT5 was in complex with MEP50  cells from The European Collection of Cell Cultures were grown as describedHepG2 cells were grown to 80% confluency, washed with ice-cold PBS, collected and centrifuged at 4 °C at 1,500 × g for 3 min. After the removal of PBS cells were homogenized in 400 μl buffer A by suspending with a 26 gauge needle 10 times using a 1 ml syringe. The lysate was vortexed for 15 sec. The efficiency of lysis was judged by trypan blue staining monitoring intact nuclei. The lysate was centrifuged at 4 °C at 16,000 × g for 1 min and the supernatant was used as cytosolic fraction. The pellet was resuspended in 200 μl buffer A and passed through the 26G needle 10 times as a washing step. After centrifugation at 4 °C at 16,000 × g for 1 min 100 μl buffer B was added to the pellet and sonicated and used as nuclear fraction.HepG2 cells were grown in 96-well plates to determine viability by MTT assay and caspase-3 activity and were carried out as detailed beforeSDS-PAGE and immunoblotting were carried out as describe previouslyHepG2 cells were grown on rat tail collagen-coated coverslips (BD Biosciences) and fixed using 4% paraformaldehyde and treated as described1-1004 and GST-MYPT1667-1004 were immobilized on anti-GST coupled CM5 sensorchipa) values determined by BIAevaluation 3.1 software.SPR-based binding studies were carried out using a Biacore 3000 instrument to monitor the interaction of MYPT1 with PRMT5. GST-MYPT1T80A in pReceiver-M11 were performed using QuickChange II XL Site-Directed Mutagenesis Kit according to the manufacturer’s instructions (Agilent Technologies). Primer pairs are listed in Expression vector pReceiver-M11 encoding wild-type human full-length PRMT5 (NM_006109.3) transcript variant 1 was purchased from GeneCopoeia Inc., USA. Point mutations of PRMT5For LC-MS/MS analysis of FT-MYPT1 interacting proteins FT-MYPT1 and Flag-peptide as control were bound to anti-Flag M2 affinity gel. The samples were precleared on the anti-Flag M2 affinity gel prior to affinity isolation. After washing with Tris-buffered saline solution (TBS) three times, beads were incubated with HepG2 nuclear extract for 2 hr on 4 °C. The FT-MYPT1 was eluted with 300 μg/ml Flag-peptide from the beads with the anchored proteins. Eluted samples were subjected to SDS-PAGE. Bands of interest were visualized by MS compatible silver staining and the proteins were subjected to in-gel digestion using trypsin. No proteins other than human keratin contamination were identified from the control samples.1-296 and -PRMT5 antibodies were incubated with Protein-A Sepharose  as described before1-296and anti-PRMT5 antibodies followed by Clean-blot IP detection reagent (HRP).For immunoprecipitation (IP) anti-MYPTFT-PRMT5wt was bound to anti-Flag M2 affinity gel following phosphorylation by protein kinases and was applied for the methylation assay. 2 μM S-adenosyl-L-methionine and 0.02 mg/ml of histone mixture from calf thymus (Roche) were added to the reaction in a total volume of 50 μl. Methylation reaction was allowed to proceed for 2 hours at 30 °C in the presence or in the absence of 5 nM recombinant PP1cδ32P-ATP or for LC-MS/MS analysis by 0.5 mM ATP in the absence (control) or in the presence of 0.4 U/ml ROK , 0.1 μg/ml PKA or 0.1 μg/ml PKC at 30 °C for 120 min. ROK was applied in buffer C and all kinase assay buffers contained 1 μM mycrocystin-LR (MC-LR). PKA and PKC assay buffer are described in Phosphorylation of recombinant human PRMT5  was initiated by 0.2 mM 2 and 1 μM MC-LR at 30 °C using FT-PRMT5wt bound to anti-Flag M2 affinity gel. After 15 min incubation kinase assay medium was removed and beads were washed by TBS. Dephosphorylation of ROK-phosphorylated FT-PRMT5wt bound to the beads (the product of the kinase assay) was initiated by adding 25 nM FT-MYPT1, 5 nM rPP1cδ or the combination of these two proteins at 30 °C for 15 min, then the beads were washed by TBS and the bound (eluted??) proteins were subjected to SDS-PAGE. Bands were detected by autoradiography or visualized by MS/MS compatible silver staining. Bands of PRMT5 were cut out and subjected to in-gel digestion using trypsin. Approximately 80% of the peptide mixtures were subjected to phosphopeptide enrichment using TiO2T80 specific antibody on Western blots and analysed by densitometry normalized to the PRMT5 input using Image J. (NIH) software.Phosphorylation reaction by ROK (0.4 U/ml) was also carried out in the absence or in the presence of 10 μM H1152 with 0.5 mM ATP, 5mM MgClTotal RNA was extracted from non-target control and MYPT1 silenced HepG2 cells using TRIzol reagent (Invitrogen) as described in the manufacturer’s protocol. NanoDrop ND-1000 spectrophotometer (Thermo Scientific) was used to determine the RNA concentration. RNA was treated with RNase free DNase I (Thermo Scientific) and reverse transcription was performed with qPCRBIO cDNA synthesis kit (PCR Biosystems). PCR amplification was carried out with Maxima Hot Start PCR Master Mix (Thermo Scientific) based on manufacturer’s protocol. RT-PCR products were analyzed by agarose gel electrophoresis. The expression level of the genes of interest were performed by Image J. 1.46, normalized to GAPDH and represented as relative numbers. The primer pairs used in the RT-PCR experiments are summarized in Expression pattern of non-target and MYPT1 silenced HepG2 cells was examined and analysed as detailed beforehttp://msf.ucsf.edu/ingel.html]. For the identification of MYPT1 interacting proteins, the resulting peptide mixtures were analyzed directly by data-dependent „triple play” LC-MS/MS using a 3D-ion trap mass spectrometer . Identification of MYPT1 interacting proteins and the PRMT5 phosphorylation sites from LC-MS/MS data were detailed in the Bands of interest were subjected to in-gel digestion using side-chain protected porcine trypsin  [ SomaPlex reverse phase protein microarray slides containing human liver and normal tissue samples from 25 clinical cases and 15 common cancer cell line lysates in triplicates were purchased from Protein Biotechnologies. Microarray slides were handled following the manufacturer’s instructions as detailed in n means the number of independently performed experiments.Normalized data were analyzed using either Student’s t-test (for two groups) or analysis of variance . Post hoc testing for one-way ANOVA was determined by Tukey’s test. Parametric statistical tests were used if the assumptions of such tests were met. All normalized variables used in statistical analyses were found to be normally distributed. Tests were conducted using GraphPad for Windows. All data presented in this work represent mean ± SEM, How to cite this article: Sipos, A. et al. Myosin phosphatase and RhoA-activated kinase modulate arginine methylation by the regulation of protein arginine methyltransferase 5 in hepatocellular carcinoma cells. Sci. Rep.7, 40590; doi: 10.1038/srep40590 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Dkk) family of Wnt antagonists interrupt Wnt-induced receptor assembly and participate in axial patterning and cell fate determination. One family member, DKK3, does not block Wnt receptor activation. Loss of Dkk3 expression in cancer is associated with hyperproliferation and dysregulated ß-catenin signaling, and ectopic expression of Dkk3 halts cancer growth. The molecular events mediating the DKK3-dependent arrest of ß-catenin-driven cell proliferation in cancer cells are unknown. Here we report the identification of a new intracellular gene product originating from the Dkk3 locus. This Dkk3b transcript originates from a second transcriptional start site located in intron 2 of the Dkk3 gene. It is essential for early mouse development and is a newly recognized regulator of ß-catenin signaling and cell proliferation. Dkk3b interrupts nuclear translocation ß-catenin by capturing cytoplasmic, unphosphorylated ß-catenin in an extra-nuclear complex with ß-TrCP. These data reveal a new regulator of one of the most studied signal transduction pathways in metazoans and provides a novel, completely untapped therapeutic target for silencing the aberrant ß-catenin signaling that drives hyperproliferation in many cancers.Members of the Dickkopf ( Dkk3 locus [in vitro [Dkk3 gene failed to provide a direct link between DKK3, the Wnt/ß-catenin signaling, and control of cell proliferation. The Dkk3tm1Cni mutant mouse is viable, fertile, shows no ß-catenin signaling defects or any increase in cancer susceptibility [The Dickkopf family of secreted glycoproteins is composed of four members that first appeared in early metazoans as key regulators of the Wnt/ß-catenin signaling pathway –4. Threek3 locus –15 and ein vitro –19. To dtibility  and failtibility –25 or detibility –32.Dkk3 gene encodes a second vital intracellular isoform, DKK3b, that inhibits hyperproliferation in cancer cells by blocking the ß-catenin nuclear translocation downstream of the Wnt-regulated ß-catenin destruction complex. In normal mouse fibroblasts, loss of DKK3b disrupts cell adhesion. This newly discovered Dkk3 gene product is an obligatory negative regulatory element in the ß-catenin signaling axis that adds a non-canonical attenuating mechanism to one of the most studied signal transduction pathways in metazoan systems. DKK3b captures ß-catenin in an extra nuclear complex with ß-TrCP preventing its nuclear translocation and serving as a gatekeeper for ß-catenin nuclear entry that modulates ß-catenin-dependent gene expression.In this study, we show that the 2 asphyxiation followed by decapitation. Frozen whole brain from homozygous and heterozygous Dkk3tm1Cni were the gift of Dr. C. Niehrs.Pregnant Sprague Dawley rats were purchased from Charles-River Labs. C57Bl/6J and CD1 mice were obtained from Jackson Labs and Charles River respectively. All rodents used in this study were maintained in an AALAC-accredited facility. The Animal Care and Use Committee of the University of Massachusetts Medical School (Assurance #A3306-01) approved the use of animals. All rodents were euthanized by CODkk3 gene (NCBI: NC_000073.6) were identified (nt8312-nt8341: AGCCCCTTTTCttcacctCAGTTGTAACTG) [5’ HA-ZFN : 2 YPYDVPDYATERPYKCPECGKSFSRSDTLKEHQRTHTGEKPYACNHZinc Finger Nuclease (ZFN) target sites within intron 2 of mouse GTAACTG) , 34 and GTAACTG) : The comPVESCDRRFSRSSHLTRHIRIHTGQKPFQCRICMRNFSRSDHLTQHIRTHTGEKPFACDICGRKFAQRGNLTRHTKIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQDYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF-COOH and the 3’ Flag-ZFN : 2-DYKDDDKTERPYKCPECGKSFSRSDTLVNHEHQRTHTGEKPYACPVESCDRRFSQRGNLTTHIRIHTGQKPFQCRICMRNFSRSDALRSHIRTHTGEKPFACDICGRKFARSDNLSEHTKIHTGGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQAREMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINF-COOH. The homologous recombination donor DNA for the mouse target Dkk3 locus was assembled by PCR amplification. PCR primers are listed in  derived from the C57Bl/6J mouse cerebellum. C8D1A cells were transfected with Dkk3 targeted ZFN plasmids and genomic DNA was isolated 48 h later. The 681 bp target locus was PCR amplified, heat denatured, re-annealed and indel formation evaluated by Cel-1 assay [5’CAGTCTTGGCACCTATAGAAGAGGGGAAGAGAAGGCAGCCCCTTTTGAATTCTTGTAACTGAAAGAAACCGTTTGTGTCTTTGATTTGATAGATG) along with the Dkk3 targeted ZFN plasmids using Fugene6. The Dkk3 target sequence was PCR amplified from gDNA isolated 48 h after transfection. HR mediated repair of the ZFN generated DSB was confirmed by the EcoRI restriction of the PCR amplified target sequence.The 824 bp 5’ homology arm (nt7415-nt8239) and 722 bp 3’homology arm (nt8342-nt9064) were appended to a loxP-TagCFP-pA-loxP cassette to create a linear 2.7 kb -1 assay  (Transge-1 assay . C8D1A cin vitro (Ambion), purified (Qiagen) and injected along with the 2.7 kb linear Dkk3b HR Donor DNA into the nucleus and cytoplasm of C57Bl/6J zygotes (UMMS Transgenic Core). Injected zygotes were implanted into the uteri of pseudo-pregnant C57Bl/6J dams and gene edited pups were identified by genotyping  was used for 1st cDNA synthesis and the 5’UTR of the Cfp mRNA was amplified from the 1st strand cDNA by 5’RACE PCR using the kit upstream UPM primer (5’-GTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT) and two, independent, Cfp-specific downstream primers each with an appended infusion cloning sequence (bolded nucleotides): one located 384 nucleotides downstream from the Cfp ATG start site CFP384R (gattacgccaagcttCTCGCCCTTCAGCTCGACGCGGTTCACC5’-) and a second located 234 nucleotides downstream of the Cfp ATG start site CFP234R (gattacgccaagcttCATGTGCTCGGGGTAGCGGGCGAAGC5’- ). Specific PCR products of ~500 bp and ~300 bp, respectively, were gel isolated, cloned into the pRACE vector (TaKaRa) and 15 clones from each Cfp primer were DNA sequenced.The transcription initiation site in exon 2 of the mouse 2 in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% bovine calf serum (BCS), 50 units/ml penicillin, 90 units/ml streptomycin. All cell cultures were passaged every 5–7 days for up to 3 passages.Primary astrocytes were prepared from one-day-old neonatal rat or mouse . Mouse e2 24 h prior to transfection.PC3, HEK293, HeLa and C8D1A cells were maintained in DMEM supplemented with 25 mM HEPES (pH 7.4) 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Cells were seeded at a density of 15,000 cells/cmTransient transfections were done with Fugene6 according to manufacturer’s instructions. Cell lysates were prepared 48 h after transfection. Each transfection experiment repeated at least 3 times and each assay point was determined in triplicate.Tunable ectopic protein(s) expression was done using the Tet-ON expression system (Life Technologies). The Tet Repressor Protein (TR) was integrated in the genome of PC3 cells by lentivirus infection  and cell selected with G418. TR expressing PC3 cells were transfected with pTREX-DKK3-HA or pTREX-Flag-IBS and ectopic protein expression induced by addition of increasing concentrations of doxycycline to the growth medium.Dkk3 gene. The Tcf promoter [Promoter analysis was done with PCR isolated segments of intron 2 of the rat promoter  . Immune complexes were eluted in 50 mM Tris, pH 8, 1 mM EDTA, 1% (v/v) SDS at 65°C for 15 min, adjusted to 200 mM NaCl, and cross-links broken by heating at 65°C for 16 h. Immune complexes were then treated with RNase I (1 unit/ml) and Proteinase K (20 μg/ml) and DNA purified (Qiagen). Immune-precipitated DNA and input DNA  were analyzed by real time PCR  using the SYBR Green PCR kit (Qiagen) and primers TSS2F and TSS2R (Chromatin immunoprecipitation (ChIP) was done as described  with modnd TSS2R . μ® Gold mountant (ThermoFisher). Tissue sections (5 μm) were prepared by the Morphology Core (UMMS) from paraffin embedded, formalin fixed organs from a Dkk3CFP/+ mutant mouse. Dewaxed sections were stained with anti-CFP antibody (1 μg/ml) and counter stained with hematoxylin and eosin. Antibodies used in this study were: anti-HA (ab1424), anti- ß-tubulin (ab6046), anti-DKK3 (ab186409) (Abcam), anti-FLAG , anti-Myc (SAB4300320) anti-ß-catenin (04–958) (Sigma), anti-CFP (cat#632381) (TaKaRa), anti-ß-TrCP (mAB #4394), anti-GAPDH (mAB #2118) , and anti-Dkk3b [488 and Alexafluor568 conjugated and HRP-conjugated secondary IgGs  were purchased from Life Technologies; RDye680CW and IRDye800CW conjugated secondary IgGs  coated coverslips and grown for 24 h prior to transfection. All transfections were done with Fugene6 according to manufacturer’s instructions. After 48 h, cells were washed with ice cold PBS, fixed with 4% paraformaldehyde in PBS for 10 minutes and quenched with 10 mM glycine. Fixed cells were permeabilized with Triton X-100, blocked with 1% BSA in TRIS buffered saline containing 0.1% Tween 20 and then incubated with primary antibodies (0.2–1 μg/ml) for 1 h at room temperature. After washing, immune complexes were visualized with secondary antibody-Alexafluor conjugates for 1 h. Nuclei were stained with DAPI. Coverslips were mounted with Prolongulin ab60, anti-DK6 pfu/ml per shRNA target) targeting the open reading frame of mouse DKK3 and Dkk3b transcripts (exons 4 and 8) , mouse ß-catenin (exons 5 and 8) , and human ß-TrCP  and a non-silencing control (RHS4348) were used. Cells were infected with lentiviral pools of shRNAs (>1 x 106 pfu/ml per shRNA target), grown for 2 days and selected with puromycin. GFP positive shRNA expressing cells were used in all experiments.MEFs were infected with pools of lentiviral shRNAs in pGIPZ from (Dharmacon) archived by UMMS RNAi Core. Lentiviral pools of shRNAs . TaqMan® Fast Advanced Master Mix and taqMan® probes for mus Dkk3 exons 1–2, Mm01269624; mus Dkk3 exons 3–4, Mm00443801; mus Cyclin D1; Mm00432359; mus c-Myc, Mm00487804; human Cyclin D1, Hs00765553; human c-Myc, Hs00153408; human Axin2, Hs00610344; human Cox2, Hs000153133; human Wfdc2, Hs00899484; human Cdh2, Hs00983056; human; human Fosl1, Hs04187685; human, Tuba, Hs0102675; and human Gapdh, Hs03929097 were purchased from Life Technologies.Total cellular RNA was isolated using Trizol Reagent (Life Technologies) or RNeasy (Qiagen) according to the manufacturer instructions. RT-PCR was performed with SuperScript First-Strand Synthesis System (Life Technologies) and primed with either Oligo(dT) or random hexamers. First strand cDNAs primed with either Oligo(dT) or random hexamers yielded identical results. Initially, real time PCR  was done with a SYBR Green PCR kit (Qiagen). All subsequent experiments were done using multiplexed TaqManCells expressing the epitope-tagged target proteins were lysed in 1X IP buffer (ThermoFisher Sci) with 150 mM NaCl, protease and phosphatase inhibitors. Lysates were incubated with anti-epitope antibody for 3 hrs at 4°C and immune complexes collected on ProteinA/G-agarose beads (Sigma). Immune complexes were washed five times with lysis buffer and eluted in Laemmli buffer containing 1% SDS. Cell extracts and immune complexes were separated by SDS–PAGE and immunoblot analysis done with anti-Flag, anti-Myc and anti-HA antibodies.® (7.2 μg/mg resin) and control rabbit IgG affinity DynaBeads® (8.4 μg/mg resin) were prepared by coupling affinity purified anti-Dkk3b IgG [™magnet. Acid eluates were then separated on 10% SDS-PAGE gels and target proteins identified by immunoblot. All native co-immunoprecipitation experiments were repeated three times.Native ß-catenin and ß-TrCP in HeLa cells were co-immunoprecipitated from cells treated for increasing times with TAT-Dkk3b. Anti-Dkk3b affinity DynaBeadskk3b IgG  and rabbDkk3b cDNA was cloned into the pTAT-HA plasmid , a gift Dkk3 knockout mouse (Dkk3tm1Cni) [The unaltered ß-catenin signaling in the 3tm1Cni)  led us t3tm1Cni) , 48. AmiDkk3tm1Cni mutant mouse was generated by replacement of the majority of exon 2 of the Dkk3 gene with an in-frame LacZ-stop cassette [The cassette . Exon 2 Dkk3 gene encodes the initiator methionine of Dkk3b from frogs to man, suggesting that Dkk3b is generated from a second Dkk3b transcript, possibly initiating within the 6 kb intron 2  to the 250 nucleotides just upstream of the 5’ end of exon 3  driven by artificial nucleases to selectively disrupt this transcript within the mouse genome. A promoter trap knock-in strategy was devised using zinc finger nucleases  (ZFNs) tced mRNA . The CFP1A cells . These mk3 locus –51. Sinck3 locus  and presk3 locus —we examipression .Dkk3b mRNAs and a linear HR donor DNA to create the Dkk3bCFP knock-in mouse. Thirty-five of 65 (54%) injected one cell embryos produced viable pups, and DNA sequencing of the target locus confirmed that 1 male (#17) and 2 females (#18 & #19) (8.6%) had HR mediated insertion of a floxed CFP decoy inserted 35 nucleotides upstream from exon 3 .Excision of the floxed CFP cassette (ΔCFP) in the unfertilized mutant oocyte using a Sox2 promoter-driven Cre recombinase , 54 rescP/+ mice  and showx vivo gene editing of heterozygous mutant MEFs was used to confirm the promoter trap knock-in strategy. Bi-allelic disruption of the Dkk3b locus was done in heterozygous mutant MEFs (Dkk3bCFP/+) using a second round of ZFN-initiated, HR repair to insert an mCherry reporter into the remaining wild type allele. Forty-eight hours after ex vivo gene editing, viable MEFs with bi-allelic mutations at the target locus expressing both fluorescent proteins were found attached to the dish, as well as free floating in the growth medium. Mutant Dkk3bCFP/mCherry MEFs, both attached and free floating, were pooled and sorted by FACS. Immunoblots of sorted cell lysates showed that the Dkk3bCFP/mCherry MEFs continued to express the ~65 kDa glycosylated DKK3 protein, while the 30 kDa DKK3b was absent , >70% of the viable Dkk3/Dkk3b KD cells were found free-floating after 3 days in culture  from the k3 locus , 56.The relationship between DKK3b and ß-catenin signaling was defined by cell proliferation and promoter-driven reporter assays in HEK293 cells with reduced endogenous DKK3b. Basal cell proliferation was unaffected by ectopic DKK3b produced either by transient transfection and by addition of the TAT-DKK3b . On the Primary and downstream promoter-luciferase reporter assays determined the effects of DKK3b on ß-catenin-driven gene expression. HEK293 cells were co-transfected with Wnt1 and promoter-driven firefly luciferase constructs paired with a control CMV-driven renilla luciferase cDNA, and then treated with TAT-DKK3b for 24 h. Wnt1 stimulated cells showed a 65-fold increase in TOPflash activity and TAT-DKK3b completely arrested expression of this canonical ß-catenin reporter . DKK3b aDkk3 gene was not identified [Yeast two hybrid screens showed that DKK3 interacted with the E3 ubiquitin protein ligase, ß-TrCP, and that this complex captured cytoplasmic dephosphorylated ß-catenin , althougentified , 18. DKKS33Yß-catenin also contained both ß-TrCP and DKK3b, while control IgG precipitates failed to capture any epitope tagged targets  failed to show any interaction between these epitope-tagged binding partners  transcripts were unaffected. These data show that both reporter and intrinsic gene expression are modulated by ability of DKK3b to control ß-catenin translocation to the cell nucleus, and that this action depends on the the ability of DKK3b to capture the native transactivator in an extranuclear complex with ß-TrCP.Since DKK3b silenced both TOPflash activity and captured the native transfactor in an inhibitory complex with DKK3b:ß-TrCP, we determined the effects of TAT-DKK3b on native gene expression in resting and LiCl-stimulated HeLa cells. Like the TOPflash assay, addition of TAT-DKK3b to unstimulated HeLa cells led to a 40–60% decrease in both native ter 16 h . Similarilencing , while eDKK3 is the enigmatic member of an ancient family of secreted glycoproteins that regulate the Wnt/ß-catenin pathway by interrupting the assembly of a functional Wnt liganded receptor , 11. It Dkk3 gene encodes a second gene product, DKK3b—a vital intracellular protein that regulates ß-catenin trafficking—provides the missing component that connects the Dkk3 gene to its regulation of the ß-catenin signaling pathway. DKK3b acts on the ß-catenin signaling pathway independent of the Wnt modulated destruction complex and modulates a diverse array of cellular functions, such as cell proliferation, cell attachment, embryogenesis and gene expression. Positioned downstream of the Wnt regulated degradation complex, DKK3b modulates ß-catenin trafficking to the nucleus. In astrocytes, DKK3b rapidly shuttles between the perinuclear space and the plasma membrane using myosin motors and actin fibers [The recognition that the n fibers , 62, sugDkk3 gene joins a growing list of mammalian genes that use multiple alternate promoters to achieve functional diversity [Dkk3 gene and the demonstration that it generates a second transcript encoding a vital intracellular isoform was unexpected. Using targeted gene editing to abolish Dkk3b expression, while preserving expression of its sister (Dkk3), we found that DKK3b was essential for early embryonic development at or near implantation, dramatically different from the Dkk3tm1Cni mutant mouse where secreted Dkk3 expression was inactivated [The iversity , 64. Ourctivated . Despitectivated , 66, actctivated , and is ctivated . Using activated  found thctivated  were resDKK3b-deficient MEFs showed both elevated ß-catenin signaling and cell attachment defect(s); two events likely to interrupt orderly development of the embryo. These disruptions in the signaling pathway were rescued by replacement with ectopic DKK3b or by simultaneous knockdown of ß-catenin. These data illustrate that the loss of DKK3b directly leads to aberrant ß-catenin signaling and disrupts cell-cell and cell-substrate interactions.Dkk3 gene is common in cancer and ectopic over-expression of DKK3 arrests tumor cell growth [Dkk3 gene, revealed that DKK3b arrests Wnt stimulated cell proliferation and selectively silences ß-catenin dependent gene expression in both immortal and cancer cells. The ability a purified cell-penetrating DKK3b protein to fully restore control of ß-catenin signaling by partnering with ß-TrCP to capture the transfactor in an extra nuclear complex offers a promising new therapeutic target for ß-catenin driven hyper-proliferative disease(s) like cancer.Unlike its ubiquitous expression in somatic cells, silencing of the l growth –19. AnalAn essential partner in the molecular mechanism of DKK3b action is ß-TrCP, an F-box protein with WD40 repeats that captures a broad range of protein targets through a consensus dephosphorylated 6 amino acid long degron domain in the target protein(s) that is located 10 to 20 residues downstream of the lysine used for ubiquitin conjugation . DKK3b cDKK3b is a novel and essential component of the Wnt/ß-catenin signaling pathway that plays a key role in both development and cancer. It serves as a gatekeeper for ß-catenin nuclear entry, directly modulates this pro-proliferative signaling molecule, and provides an important new point of control that impacts the regulatory pathways responsible for differentiation, lineage specification, pluripotency and oncogenesis.S1 FigDkk3 cds. (B) Validation of the Dkk3 exon 2 and exon 3 primer sets using increasing concentrations of 1st strand cDNA primed total RNA isolated from two independent mouse astrocyte preparations. Each data point determined in triplicate. (C) Dkk3 mRNA levels were normalized to GAPDH mRNA. Data shown as (mean ± SE) from 3 independent experiments.(A) The position of the PCR primer pairs used for amplification of exon 2 and exon 3 shown on the (TIF)Click here for additional data file.S2 FigDkk3 gene. TSS2 is positioned upstream of the forward LoxP site (black box) of the gene and the downstream LoxP site (in black) is positions 35 nt upstream of exon 3. Position of the CFP234 5’RACE primer indicated by arrow. (B) Sequence of the 5’UTR of the Cfp mRNA captured by 5’RACE highlighted in yellow.(A) Map of the insertion of the CFP promoter trap in gene-edited intron 2 of the (TIF)Click here for additional data file.S3 FigCyclinD1 and Myc expression in HeLa cells ±TAT-DKK3b for 16 h. (B) Native ß-catenin dependent gene expression in HeLa cells. Cells were stimulated with ±LiCl in the absence or presence of TAT-DKK3b for 16 h. QPCR data are reported as % of unstimulated controls for each target transcript and expressed as means ± se, n = 9. Gene products probed: Cdh2, N-cadherin; Axin2, Axin2; Cox2, cyclooxygenase-2; Wfdc2, WAP Four-disulfide Core Domain 2; Fosl1, FOS Like Antigen 1; Tuba, alpha tubulin.(A) Basal (TIF)Click here for additional data file.


Editorial on the Research TopicCross-Talk Mechanisms of Wnt/Beta-Catenin Signaling Components with TLR-Activated Signaling Molecules in the Inflammatory ResponseMolecules and signaling pathways involved in an inflammatory response have been subjects of numerous original and review articles in the last decade. At the beginning and for many years, scientists were mainly interested in deciphering the intricate events involved in triggering and maintaining inflammation. Such studies led to the establishment of NF-κB as one of the main transcription factors that regulates the expression of functionally related genes  involved in controlling inflammation, cell survival, and cancer .Mycobacterium.An uncontrolled cell proliferation and the tumor microenvironment characterized by cells secreting pro-inflammatory cytokines, mostly promoted by NF-κB activity, are undoubtedly the most characteristic hallmarks of all types of cancers . During Ma and Hottiger is a very well detailed overview of the different molecules and mechanisms that reciprocally regulate the activities of NF-κB and Wnt/beta-catenin. The authors start with a description of the canonical NF-κB and Wnt/beta-catenin signaling pathway and then concentrate the discussion on the alternative mechanisms used by NF-κB to positively or negatively control the transcriptional activity of beta-catenin and the mechanisms used by beta-catenin to positively or negatively modulate the activity of NF-κB in inflammation, and inflammation-associated diseases such as cancer. Importantly, they conclude that positive or negative control of one signaling pathway over the other is cell and context-dependent and propose that new therapies against cancer should be focused on controlling inflammation and cell proliferation simultaneously.The work by Zolezzi and Inestrosa gives a clear account on the possible interrelationship between pro-inflammatory genes expressed by the TLR/NF-κB and Wnt/beta-catenin signaling pathways in the AD. These authors point out that the production of proinflammatory cytokines, like IL-1β, IL-6, IL-12, TNFα, the enzymes cyclooxygenase 2, and the inducible nitric oxide synthase are likely mediated by astrocytes, microglia, neurons, and oligodendrocytes. Interestingly, in a normal brain, the synthesis of these proinflammatory factors is counterbalanced by the expression of anti-inflammatory genes, such as TGFβ and IL-10; however, in AD, proinflammatory gene expression is predominant since Aβ causes a chronic inflammatory condition. This unbalance is perhaps due to the failure of the anti-inflammatory control exerted by the canonical Wnt/beta-catenin components on the TLR-dependent NF-κB activation as it is discussed in the review.The AD is a neuroinflammatory disorder of the central nervous system in which the β-amyloid peptide (Aβ) forms aggregates. The article review by Suryawanshi et al. discusses the current knowledge on the function of Wnt/beta-catenin signaling in the induction of immune tolerance in homeostatic tissues, autoimmunity, and oncogenesis. An important conclusion drawn by the authors, after discussing many reports on DC roles in different diseases, such as autoimmune encephalomyelitis, rheumatoid arthritis, psoriasis, cancer, and others, is that inhibition of Wnt/beta-catenin activity induces an important reduction of inflammation. In particular, they point out that in the central nervous system a sustained activation of this pathway restrains inflammation, stimulates neuroprotection, and promotes neurogeneration. Finally, but not less important, a clear picture of the tolerogenic role of Wnt/beta-catenin in the gut through a modulatory effects of DCs is also provided.During the last few years, a number of studies have highlighted the role of dendritic cells (DCs) in the regulation of immune responses in various pathological conditions. The review by Mycobacterium tuberculosis, an intracellular pathogenic Gram-positive bacterium, has been a major burden of humanity with millions of cases per year, being one of the leading cause of death worldwide. The review by Brandenburg and Reiling, puts in perspective the major advances in our understanding of various Wnt  and Frizzled (FZD1) genes function, the upregulation of FZD1 by IFNγ, and the relationship of Wnt/beta-catenin with TLR (TLR2 and TLR4) signaling during M. tuberculosis lung infection. Most importantly, they discussed the major contribution of these molecular components inside the granuloma and surrounding tissues.An increasing number of reports has documented the modulation of Wnt/beta-catenin signaling in different bacterial infections . InfectiVillaseñor et al. on the interconnection between Wnt/beta-catenin and M. tuberculosis. These authors emphasize that M. tuberculosis induces the secretion of proinflammatory cytokines  from macrophages at early stages of the infection, while at later stages, anti-inflammatory cytokine  expression predominates, which contributes to mycobacterium survival and granuloma formation. Moreover, in addition to a detailed account of Wnt3, Wnt5, and Wnt6 on mycobacterium infection, a noteworthy mention on NOD2 and a variety of miRNAs is included, giving a wider picture of the regulatory mechanisms developed in infections caused by M. tuberculosis.Another excellent review included in this special issue is the review of In light of the current experimental evidence on the molecular programs that control an inflammatory response, one would expect major breakthroughs based on the interrelation and integration of signaling pathways such as canonical TLR-IKK-NF-κB and Wnt/beta-catenin. A deep understanding of the regulatory mechanisms among these signaling pathways will be critical to design drugs aimed at effectively controlling inflammation by affecting different protein interactions that are fundamentally altered in chronic and degenerative diseases.VMB-A edited all the article reviews and wrote the editorial of the Research Topic.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The Kd, Bmax, internalization and binding kinetics of Ex-4 and Ex(9–39) were studied in vitro using Ins-1E cells. Biodistribution and imaging studies were performed in nude mice bearing Ins-1E xenografts. In vitro evaluation demonstrated high affinity binding of the Ex-4 agonist to the Ins-1E cells with fast internalization kinetics reaching a plateau after 30 min. The antagonist Ex(9–39) did not internalize and had a 4–fold higher Kd value compared to the agonist. In contrast to Ex(9–39), which showed low and transient tumor uptake, Ex-4 demonstrated excellent in vivo binding properties with tumor uptake identical to that of 68Ga-Ex-4, but substantially lower kidney uptake resulting in a tumor-to-kidney ratio of 9.7 at 1 h compared to 0.3 with 68Ga-Ex-4. Accumulation of activity in thyroid and stomach for both peptides, which was effectively blocked by irenat, confirms that in vivo deiodination is the mechanism behind the low kidney retention of iodinated peptides. The 124I congener of Ex-4 demonstrated a similar favourable biodistribution profile in the PET imaging studies in contrast to the typical biodistribution pattern of Ex-4. Our results demonstrate that iodinated Ex-4 is a very promising tracer for imaging of benign insulinomas. It solves the problem of high kidney uptake of the radiometal-labelled tracers by improving the tumor-to-kidney ratio measured for Ex-4 by 32 fold.GLP-1 receptors are ideal targets for preoperative imaging of benign insulinoma and for quantifying the beta cell mass. The existing clinical tracers targeting GLP-1R are all agonists with low specific activity and very high kidney uptake. In order to solve those issues we evaluated GLP-1R agonist Ex-4 and antagonist Ex(9–39) radioiodinated at Tyr Glucagon like peptide-1 receptors (GLP-1R) are ideal targets for the imaging of benign insulinoma due to their high density on the surface of more than 90% of these tumors ,3. Since111In Ex-4.However, the Bolton-Hunter method is not so trivial to translate into the clinic. In the current study we selected the direct iodination approach, because it is a one-step process and the  125I-BH . As a coAll reagents and solvents were purchased from Sigma-Aldrich  and Carl Roth GmbH & Co. KG . The details for the analytical and semi-preparative HPLC set up are described in the 14,Tyr40-NH2]Ex-4 and Ex-(9–39) precursor peptides were purchased from Biotrend Chemikalien GmbH . Ex-4 and Ex(9–39) were produced from precursor peptides by Celerion  using the Chloramine-T method. Ex-4 and Ex(9–39), the corresponding non-radioactive reference peptides were custom-synthesized by Peptide Specialty Laboratories GmbH  and Biotrend Chemikalien GmbH , respectively, using a Fmoc-protected 3-iodotyrosine as a building block.Ex-4 was prepared from Ex-4 using I-124  carrier-free , employing the Chloramine-T method following a published protocol Ex-4 and increasing amounts of Ex-4 peptide (1–100 nM) in the growth medium, containing 1%FBS for 2 hours at +4°C. To determine the non-specific binding, three additional wells were blocked with 1 μM of Ex-4. After washing with cold PBS cell bound activity was collected with 1M NaOH (3x1 ml). For internalization experiments, Ins-1E cells were incubated with 5 kBq of Ex-4, containing 1 nM of Ex-4 at 37°C. At specific time points the internalization was stopped by washing the cells with ice-cold PBS and the membrane bound and internalized activity were collected as described previously Ex-4 or Ex(9–39) in 100 μL sterile saline via intravenous (i.v.) tail-vein injection. For mass dependence 10 or 100 pmol of Ex-4 or 10 pmol of Ex(9–39) were coinjected together with the radioiodinated peptide. For the blocking experiment, 80 nmol of Ex(9–39) was injected 5 minutes before the administration of iodinated peptide. The sodium iodide symporters were blocked by Irenat (Bayer) . Animals  were euthanized by asphyxiation with excess isoflurane at 1, 4 and 24 h post-radiotracer administration, and tissues were removed, rinsed in water, dried in air, weighed and counted on a calibrated and normalized gamma-counter.Ins-1E tumor-bearing mice were administered with 40 kBq of Ex-4 . At 1, 2 and 4 hour post injection 20–40 minute static scans were acquired using microPET Focus 120 scanner (Concorde Microsystems), followed by 2 minute CT scans on micro-CT-Tomoscope Synergy (CT Imaging GmbH). PET sinograms were reconstructed using a 2-dimensional ordered-subset expectation maximization (2D-OSEM) algorithm. Image counts per pixel per second were calibrated to activity concentrations (Bq/mL) by measuring a 3.5 cm cylinder phantom filled with a known concentration of radioactivity. The PET and CT images were co-registered using the Rover software . PET images were analyzed using Rover software . The regions of interest were drawn based on the CT to include the entire tumor or the kidney and the mean %IA/g was calculated. For the time activity curves the PET images recorded at 1, 2 and 4 h p.i. were merged together and the mean %IA/g were calculated from the VOI placed in the same position in the tumor.Mice were injected t-test.A statistical analysis was performed using GraphPad Prism 5.01  and Microsoft Excel. Data were analyzed by using the unpaired, two-tailed Student’s 14,Tyr40-NH2]Ex-4 and Ex(9–39), and the non-radioactive reference peptides,Ex-4 and Ex(9–39) was determined by analytical HPLC to be >95%. Compounds were characterized by either ESI-MS or MALDI-MS and the obtained masses matched the exact calculated masses for all compounds  were delivered carrier-free, with a specific activity of 81.4 TBq/mmol and a radiochemical purity of > 95%  antagonist were studied using Ins-1E cells, expressing the rat GLP-1 receptor. The results of the saturation binding experiments demonstrate that Ex-4 bound to Ins-1E cells with a Kd of 4.1±1.1 nM and a Bmax of 0.045±0.002 nM , in contrast, had a Kd value of 18.1±6.9 nM and a Bmax value of 0.071±0.007 nM  in Ins-1E cells. The amount of internalized Ex-4 after 0.5 h at 37°C was 3.12±0.11% reaching 3.75±0.06% after 4 h  showed very different kinetics with a significantly higher percentage of specifically bound peptide (1.82±0.09%) and a lower amount of internalized peptide (0.52±0.05%) at 0.5 h. After 4 h the amount of bound peptide remained the same  followed by the lung (30.5±3.6%IA/g) and the pancreas (25.3±4.2%IA/g) (At 1 hour post injection (p.i.) of Ex-4 1.4±0.1%IA/organ was accumulated in the thyroid, confirming the dehalogenation of the tracer in vivo. However, blocking the sodium iodide symporter with irenat reduced the thyroid uptake by 94%  the lowest peptide mass of 0.5 pmol corresponding to the highest specific activity showed the most favourable biodistribution profile with highest tumor uptake and highest tumor-to-normal organ ratios  was lower, 12.7±4.1%IA/g at 1 h p.i. and it decreased after 4 h to 1.9±0.5%IA/g, similar to the activity in the blood pool  were substantially lower than the ratios for the agonist. The uptake in the thyroid was 1.3±0.2%IA/organ at 1 h p.i., indicating tracer deiodination in vivo. In terms of the peptide dose, the lowest dose of 0.5 pmol yielded the highest tumor uptake and best tumor-to-normal organ ratios NH2]Ex-4, the kidney had the highest accumulation of activity (201.3±30.6%IA/g) followed by the Ins-1E tumor (58.3±15.6% IA/g), the pancreas (25.5±5.2% IA/g), and the lung (14.3±1.2%IA/g). The tumor-to-blood and tumor-to-muscle ratios were 48.5 and 215.9, respectively  radiochemical yield and >95% radiochemical purity  PET images (st panel) demonstrate that at 1 hour p.i. of Ex-4, the highest specific uptake was detected in Ins-1E tumor (10.8%IA/g based on image quantification). In the blocked mouse (nd panel) the tumor uptake was inhibited by 82% (1.9%IA/g left in the tumor). Apart from the bladder, high accumulation of radioactivity was also found in the thyroid (19.7%IA/g) and the stomach (28.3%IA/g) confirming the tracer radiodeiodination in vivo. The stomach uptake was partially inhibited in the blocked mouse, suggesting some GLP-1 receptor mediated uptake in the stomach, which was also described by others in the field. Blocking of the sodium iodide symporter with irenat did not affect the tumor uptake (11.4%IA/g) but completely inhibited the thyroid uptake and most of the stomach uptake (rd panel).The 14,124I-Tyr40-NH2]Ex-4 revealed that there was only free 124I-iodide and no intact peptide present in the mouse urine NH2]Ex-4 shows a decrease in the tumor radioactivity from 11.4%IA/g at 1 h p.i. to 5.4%IA/g at 4h p.i. NH2]Ex-4 at 1 h p.i .14,125I-Tyr40-NH2]Ex-4 showed high affinity binding to Ins-1E cells in vitro with a Kd of 4.1±1.1 nM. The Kd value for Ex(9–39), however was 4.4 fold higher, demonstrating a significantly lower affinity towards GLP-1R. In contrast to the radiometal-labelled GLP-1R tracers, the fraction of internalized Ex-4 did not increase with time after reaching the maximum of 3% at 0.5 h. This phenomenon has been previously described for radioiodinated tracers Ex–39, howeabolites . As a re14,125I-Tyr40-NH2]Ex(9–39) behaved in in vitro binding assays as expected from an antagonist. In comparison to the agonist Ex-4, it showed higher cell membrane binding but a 5.5-fold-lower internalization into Ins-1E cells. Interestingly, the Bmax value for the iodinated antagonist was higher , potentially indicating that it recognizes more binding sites than the iodinated agonist. This feature of the antagonist tracer has been described for the SST-2 receptors, where antagonists recognized 10–15 fold more binding sites, compared to the agonists Ex-4 demonstrated high and specific tumor uptake, which was equal to the tumor uptake of 68Ga-Ex-4. But most importantly the kidney uptake was very low resulting in the high tumor-to-kidney ratio of 9.7. This ratio was even better than the ratio of 3.5, which we reported for the antagonist 125I-BH-Ex(9–39) Ex-4 and 68Ga-Ex-4, which would translate into similar imaging contrast for insulinomas against the normal pancreas.Ex(9–39) , and was14,125I-Tyr40-NH2]Ex(9–39) was significantly lower, only 12.7±4.05%IA/g at 1 h p.i. and the activity was completely washed out after 4 h p.i. This may be explained by the higher Kd value of the compound. The kidney uptake of the iodinated antagonist was identical to the agonist , but the tumor-to-kidney ratio and other tumor-to-normal organ ratios were much lower.Somewhat surprisingly, the tumor uptake of the antagonist Ex-4 and Ex(9–39) (7.5%IA/g) at 1 h p.i. was even lower than that of the Bolton-Hunter-labelled analogue 125I-BH-Ex(9–39) (12.1±1.4%IA/g) Ex-4 and Ex(9–39) in comparison to 125I-BH-Ex(9–39) Ex-4, was evaluated in PET-studies. Although absolute tumor uptake levels were somewhat lower than expected, the PET-images confirmed the very favourable biodistribution profile of the 124I congener in comparison to 68Ga-Ex-4, with a pronounced and specific tumor uptake and low kidney retention. We strongly believe in the potential of 124I- and 123I-labeled Ex-4 derivatives for GLP-1 targeted imaging and currently develop automated labelling strategies. The goal is production of these tracers in high radiochemical purity, specific activity and improved radiochemical yields, which is an important factor given the high cost of 123I and 124I in particular, and the activities of these tracers needed for comprehensive imaging studies in animals and finally for clinical translation.As a proof-of-concept, the 14,125I-Tyr40NH2]Ex(9–39) recognized more binding sites on GLP-1R, it had a lower affinity than the agonist and it may not be suitable for clinical translation. The Ex-4 agonist, in contrast, showed excellent tumor uptake and at the same time exhibited pharmacokinetics superior to all other GLP-1R tracers presently available, with particularly high tumor-to-kidney ratio and good contrast to normal organs. Preclinical PET imaging data strongly suggest that Ex-4 radioiodinated with 124I may be a promising alternative to the radio-metal labelled derivatives for imaging of GLP-1 receptor positive insulinoma. It can further improve the sensitivity of the preoperative localization of benign insulinoma.Even though the antagonist Ex-4 without (A) and with co-injection of cold reference Ex-4 (B). UV-and radio-detectors were in series, resulting in a lag time of about 15 sec for the radiotrace. Numbers in the chromatograms refer to peak retention time in minutes.(PDF)Click here for additional data file.S2 FigAnalytical HPLC chromatogram of Ex-(9–39) without (A) and with co-injection of cold reference Ex(9–39) (B). UV-and radio-detectors were in series, resulting in a lag time of about 15 sec for the radiotrace. Numbers in the chromatograms refer to peak retention time in minutes. The peak at 13.88 min originates from bovine serum albumin (BSA) employed for formulation of the radiotracer.(PDF)Click here for additional data file.S3 Fig14,Tyr40-NH2]Ex-4 elutes about 3 min earlier than the iodinated product Ex-4, enabling isolation of the pure radio-iodinated tracer in high specific activity.UV-and radio-detectors were in series, with the radio-detector preceding the UV-detector. Numbers in the chromatograms refer to peak retention time in minutes. The product peak was collected in several fractions (based on the radio-trace and before entering the UV-detector), and fractions containing the pure product  were pooled, trapped on a C8-SepPak and formulated in EtOH. Note that the peptide precursor Ex-4. Note that apart from 124I-iodide (peak at 2.38 min), neither the intact tracer Click here for additional data file.S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Activation of β-Catenin by post-translational modification has been extensively studied. However, mechanism(s) of post-transcriptional regulation of β-Catenin are not well defined. In this study, we report an RNA recognition motif-containing protein  which regulates the degradation of β-Catenin mRNA. Our results show that Rbm46 is distributed primarily in the cytoplasm of mouse ESCs (mESCs) and is elevated during the process of ESC differentiation. In addition, overexpression of Rbm46 results in differentiation of mESCs into trophectoderm, while knock-down of Rbm46 leads to mESC differentiation into endoderm. β-Catenin, a key effector in the Wnt pathway which has been reported to play a significant role in the regulation of ESC differentiation, is post-transcriptionally regulated by Rbm46. Our study reveals Rbm46 plays a novel role in the regulation of ESC differentiation.Embryonic stem cells (ESCs) are pluripotent cells and have the capability for differentiation into any of the three embryonic germ layers. The Wnt/ Mouse ESCs (mESCs) were first isolated from the inner cell masses of late blastocysts . These pβ-Catenin mRNA, thereby promoting the transcription of Oct4, Rex1 and Nanog and ultimate change in ESC pluripotency [β-Catenin is also regulated by mechanisms independent of the Wnt pathway in the course of ESC development such as post-transcriptional regulation.Aberration of this transcriptional regulatory circuity, especially the aberration of the Wnt/ β-Catenin/Oct4 pathway may lead to changes in the state of ESC pluripotency which results in an increased tendency for differentiation. Oct4 is involved in the regulation of cell growth and differentiation in a wide variety of tissues and is typically expressed in pluripotent cells of developing embryos . Genome-ipotency –16. ThesIn this study, we report a RNA recognition motif (RRM) containing protein  referred to as RBM46 which is highly expressed in hESCs. RRM is one of the most abundant protein domains in eukaryotes and is involved in all post-transcriptional events including pre-mRNA processing, splicing, and mRNA editing . Some ofwww.hyclone.com), 100 mM 2- mercaptoethanol , nonessential amino acids (Gibco), 2 mM l-glutamine (Chemicon), 1 mM sodium pyruvate (Sigma), and 100 U/mL leukemia inhibitory factor (LIF). Plates were fixed and stained for alkaline phosphatase  according to the manufacturer’s protocol.E14Tg2a mouse ESCs were cultured in the DMEM supplemented with 15% (v/v) fetal calf serum  containing protease inhibitors (Roche). Western blot was performed by standard procedures; primary antibodies used in this study: anti-Oct4 , anti-Rbm46 , beta-Actin , Pabpc1 , GSK-3 , SSEA-1. Proteins were visualized with an Odyssey Two-Color Infrared Imaging System (LI-COR Biosciences) according to the manufacturer’s instructions. For Flag immunoprecipitaion, Flag M2 beads  were utilized according to the manufacturer’s protocal.5’-ATCGCG CTCGAGATGGACTACAAGGACGACGATGACAAGATGAATGAAGAAAACACTGATGGTAC-3’ and 5’-ATATG CGGCCGCTCAGAAGAATGAGGCCTGATTG-3’. Nucleotides targeting the firefly luciferase gene, which has no homology with mouse transcriptome, served as control short hairpin RNA (shRNA). The RNAi oligonucle- otides sequences used are as follows: Rbm46 (siRbm46): 5’- TCCAACAATATCACTTGCTATTCATAGAGATAGCAAGTGATATTGTTGGTTTTTTC-3’. All constructs were verified by sequencing. To generate overexpression or knock- down cells, E14Tg2a ESCs were transfected separately with pPyCAGIP or shRNA constructs using Lipofectamine 2000 and 1 mg/mL of puromycin was added at 48 h post- transfection. About 10 days later, stably transfected cells were expanded and then stored for further analysis.Full-length Mouse Rbm46 coding sequence was amplified from mouse testis mRNA and cloned into the pPyCAGIP vector with the following primers: 6 cells/ml in phosphate-buffered saline (PBS). A total of 100μl of the cell suspension (1 × 106 cells) was subcutaneously injected into the inside of the lower limbs of nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. For pPE-E14 and pPy-RBM46-E14 cell lines, seven mice were injected per cell line. For Non silence-pPE-E14 and Rbm46 shRNA-pPE-E14 cell lines, nine mice were injected per cell line. The transplanted animals were observed routinely every 3–5 days, and tumour growth was measured with a calliper every 3–5 days. The health condition of the mice were monitored and abnormal change in temperature, respiration, feeding behaviour and weight loss was considered as unhealthy signs and the mice would be euthanized. Carbon dioxide was utilized for euthanasia for all the animals. In this study, no mice became ill or died before the experimental endpoint. After 6 weeks post the transplantation, tumours were isolated from the mice. Animals were humanely sacrificed prior to teratocarcinoma isolation. The sacrificed animals were subjected to general inspection. The teratocarcinoma samples were then weighed and subjected to RT-PCR analysis. All mice used in the study were bred and maintained at the Experimental Animal Center, Peking University Health Sciences Center . The animal experiment protocol was approved by the Biomedical Research Ethics Committee of Peking University and strictly adhered to the American Physiological Society’s “Guiding Principles in the Care and Use of Vertebrate Animals in Research and Training”.For teratocarcinoma formation assay, pPE-E14, pPy-RBM46-E14, Non silence-pPE-E14, Rbm46 shRNA-pPE-E14 cells were cultured and suspended at 1 x 102, 2 mM ethylene glycol tetraacetic acid, 10 mM NaF, and 1 mM Na3VO4) containing protease inhibitors (Roche). Followed by a FLAG immunoprecipitation using FLAG M2 beads . The beads were washed and eluted with 0.1M Glycine, pH 3.0 and neutralized with 1M Tris-HCl, pH 8.0. The eluate was used for RNA extraction using Trizole reagent (Invitrogen), followed by RT-PCR to detect beta-catenin and gapdh. Primers are listed in For the RNA-IP assay, empty vector of FLAG-Rbm46 was transfected into E14 cells for 48 hours. The cells were collected and subsequently lysed with lysis buffer . Reverse transcriptase (Promega) was utilized for cDNA synthesis. Real-time polymerase chain reaction (PCR) was carried out on the SYBR Premix Ex Taq II detection system (Takara) using SYBR dye. The amount of mRNA level was determined by the ΔΔCt method. Primers are listed in Cells on coverslips were fixed in cold methanol for 15min. And were then washed in phosphate-buffered saline (PBS) for three times, cells were subsequently blocked in PBS containing 5% BSA for 30min and then incubated with mouse or rabbit primary antibodies in PBS containing 2.5% BSA for 2–12 h at 37°C. After three washes, cells were incubated with fluorescein isothiocyanate-conjugated a-mouse and PE- conjugated a-rabbit secondary antibodies for 1 h at 37°C and then with 4, 6-diamidino-2-phenylindole (Roche) for 15 min. The coverslips were washed extensively and mounted onto slides. Imaging of the cells was carried out using the Zeiss LSM 510 Meta Confocal Microscope.One million cultured cells per sample were dissociated with 0.25% trypsin . Cells were then resuspended in 1:3 volumes of ice-cold PBS and 100% ethanol and then incubated on ice for 30 min. After centrifugation, the cell pellet was washed 3 times with PBS and centrifuged for 5 min. Cells were then incubated in the dark in 1 mL PBS containing RNase  for 30 min and propidium iodide  and analyzed by flow cytometry. Analyses were performed by flow cytometry  using CellQuest software (BD Biosciences) and the ModFit software (Verity Software House).The statistical significance of the mean differences between two samples was analysed by the Student two-tailed t test. To compare means of more than three samples, One-way ANOVA with Tukey’s post hoc test was used for analysis. Data are shown as means ± SD of the results of at least three independent experiments. Values of p < 0.05 were considered significant.In our preliminary study, we found that RBM46 is highly expressed in hESC (data not shown). Given that RRM proteins are essential for early embryo development , we hypoOct4 and Rbm46 were evaluated with qPCR. Oct4 mRNA levels were significantly decreased  staining analysis to detect the undifferentiated ESCs and found that pPE-E14 cells were almost 100% AKP positive , and cloNestin, Glast, Notch, Neurogenin and Pax6), mesoderm  or endoderm , overexpression of Rbm46 significantly elevated the mRNA level of the trophectoderm markers  (Cdx2 and Eomes)  . RT-PCR d Eomes) . These rRbm46 shRNA stable cell line that can effectively reduce the mRNA and protein level of Rbm46 (Rbm46 shRNA group (65.05% vs 49.82) , while mRNA levels for markers of ectoderm , mesoderm  and trophectoderm  were unchanged  (We next investigated the influence of Rbm46 knock-down on mESC differentiation. We first established the of Rbm46 . Knock-dof Rbm46 . AKP staof Rbm46 . In addis 49.82) , and terol cells . These rnchanged . RT-PCR d Lama1) . Our datOct4 and Rex1 [Rbm46 RNA levels are inversely correlated with Oct4 RNA levels during mESC differentiation, together with the fact that overexpression of Rbm46 or knock-down of Rbm46 results in differentiation of mESCs into trophectoderm or endoderm respectively, we reasoned that Rbm46 may regulate mESC differentiation by acting on components of the Wnt pathway.The Wnt- β-Catenin pathway is closely associated with maintenance of ESC pluripotency. Previous studies have shown that activation of the Wnt pathway stabilizes β-Catenin in the cytosol by releasing it from the destruction complex. Upon release, β-Catenin can translocate to the nucleus and activate the transcription of and Rex1 , 22–24. and Rex1 , 25, 26.We first evaluated the influence of Rbm46 on the protein level of activated β-Catenin. Western blot showed that overexpression of Rbm46 significantly reduces both the protein and mRNA levels of total β-Catenin, activated β-Catenin, and Oct4 , while kβ-Catenin 3’ UTR polyA tail into the pGL3-promotor plasmid between the luciferase gene and SV40 polyA sequence (pGL3pro-3’UTR) (β-Catenin 3’ UTR polyA in a dose-dependent manner by luciferase assay (β-Catenin mRNA.The RNA binding function of Rbm46 and its location in the cytoplasm  indicateo-3’UTR)  and perfse assay , and thise assay . These dβ-Catenin mRNA could also be pulled down by Rbm46  which is a critical component of the P-bodies, is essential for mRNA degradation through the 3’ UTR polyA tail [β-Catenin mRNA, and co-focal analysis showed that Rbm46 co-localizes with Pabpc1 in the cytoplasm  analysis. MS results showed that Rbm46 interacts with multiple proteins  which are critical effectors of post-transcriptional regulation . In addiby Rbm46 , suggestlyA tail , 29. We ytoplasm . Consist are highly expressed in ESCs as compared with differentiated cells . Some RBnslation , and we β-Catenin mRNA stability and stem cell pluripotency. Our results reveal that Rbm46 is one of the important regulators of ESC pluripotency.The study reveals novel functions of Rbm46 and demonstrates the mechanism through which Rbm46 regulates S1 FigRbm46 is highly conserved among different species. (B)Schematic figure of the RRM domains of Rbm46.(A) The sequence of (TIF)Click here for additional data file.S2 FigImmunofluorescence staining shows the localization of endogenous Rbm46 and ectopic Rbm46 (Flag-Rbm46) using antibodies against Rbm46 and Flag, respectively.(TIF)Click here for additional data file.S3 FigSchematic illustration of the construction of pGL3pro-3’UTR reporter plasmid.(TIF)Click here for additional data file.S4 FigpPE-E14 cells (Mock cells) and pPy-Rbm26 cells were lysed and subjected to Flag immunoprecipitation, followed by western blot with the indicated antibodies.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.