CTNNB1). Five individual CTNNB1 gene disrupted clones (BxPC3ΔCTNNB1) were established from a BxPC-3 founder cell line. Despite the complete absence of β-catenin, all clones displayed normal cell cycle distribution profiles, overall normal morphology and no elevated levels of apoptosis although increased doubling times were observed in three of the five BxPC3ΔCTNNB1 clones. This confirms that WNT/β-catenin signaling is not mandatory for long term cell growth and survival in BxPC-3 cells. Despite a normal morphology of the β-catenin deficient cell lines, quantitative proteomic analysis combined with pathway analysis showed a significant down regulation of proteins implied in cell adhesion combined with an up-regulation of plakoglobin. Treatment of BxPC3ΔCTNNB1 cell lines with siRNA for plakoglobin induced morphological changes compatible with a deficiency in the formation of functional cell to cell contacts. In addition, a re-localization of E-cadherin from membranous in untreated to accumulation in cytoplasmatic puncta in plakoglobin siRNA treated BxPC3ΔCTNNB1 cells was observed. In conclusion we describe in β-catenin deficient BxPC-3 cells a rescue function for plakoglobin on cell to cell contacts and maintaining the localization of E-cadherin at the cellular surface, but not on canonical WNT signaling as measured by TFC/LEF mediated transcription.Pancreatic adenocarcinoma (PA) is among the most aggressive human tumors with an overall 5-year survival rate of <5% and available treatments are only minimal effective. WNT/β-catenin signaling has been identified as one of 12 core signaling pathways that are commonly mutated in PA. To obtain more insight into the role of WNT/β-catenin signaling in PA we established human PA cell lines that are deficient of the central canonical WNT signaling protein β-catenin by using zinc-finger nuclease (ZFN) mediated targeted genomic disruption in the β-catenin gene ( KRAS, CDKN2A, TP53 and SMAD4), PA is a highly heterogenetic disease Pancreatic adenocarcinoma (PA) is the most common type of cancers in the pancreas and is the fourth leading cause of cancer deaths in developed countries WNT/β-catenin signaling is one of the pathways that were identified as a core signaling pathway that is altered in most pancreatic cancers APC, AXIN1 and CTNNB1 are rare in human PA Although the WNT/β-catenin signaling pathway has been identified as one of the key pathways commonly mutated in PA, the complex role of β-catenin mediated signaling in PA has been unclear. In mouse models constitutive activation of WNT signaling is unable to initiate PA CTNNB1). While siRNA knockdown will reduce the protein load of a cell, leaving room for residual biological activity of a targeted protein, a targeted knockout allows eliminating the biological impact of residual amounts of a protein.In this study we investigated the consequence of a complete β-catenin depletion in PA by using zinc-finger nucleases (ZFNs) to generate cell lines in which β-catenin is absent due to targeted genomic disruption of the β-catenin gene . Plakoglobin localizes at the cell membranes where it interacts with E-cadherin in a similar way as β-catenin, thus indicating a functional substitution for β-catenin at the adherens junctions. Only when in addition to a β-catenin knockout, also levels of plakoglobin were reduced by small interfering RNA (siRNA), cells changed their shape and displayed a rounded morphology with an apparent disability to form normal cell to cell connections. Analysis of core adherens junction proteins in the β-catenin and plakoglobin deficient cells revealed a significant reduction of α-catenin and p120-catenin. In addition, the localization of E-cadherin in the double β-catenin and plakoglobin deficient cells was changed from being predominantly membranous to being localized in intracellular puncta. The data from the β-catenin deficient BxPC-3 PA cells points towards a central role of β-catenin in enabling cell-cell contacts.2.The human pancreatic adenocarcinoma BxPC-3 (ATCC CRL-1687) epithelial cell line was grown in RPMI-1640  supplemented with 10% fetal bovine serum, 1% penicillin/Streptomycin and 0.002 x Insulin-Transferrin-Selenium . PANC-03.27 (ATCC CRL-1469) was grown in the same media as BxPC-3 cells apart from being supplemented with 0.1 x Insulin-Transferrin-Selenium (Life Technologies). PANC-1  were grown in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/Streptomycin. All cells were propagated at 37°C in a humidified atmosphere containing 5% COCTNNB1) were established using CompoZr custom Zinc Finger Nucleases (ZFNs) (Sigma-Aldrich). In this technology, distinct ZFNs are engineered to introduce site specific genomic double strand breaks (DSBs) that would stimulate recombination processes resulting in introduction of small genomic changes at the target site CTNNB1 gene  for 10 minutes at room temperature (RT) and blocked , 5% goat serum, 0.1% Tween-20 in phosphate buffered saline (PBS)) for 1 hour at RT followed by incubation with primary antibody (Ab)  at 4°C over night. After washing in PBS cells were incubated with fluorescently labeled secondary Ab  for 30 minutes at RT. For nuclear counterstaining the cells were incubated with DAPI (1 µg/ml) for 10 minutes at RT. Cells stained on cover slips were mounted on slides using Fluorescence Mounting Medium . The following primary Abs and dilutions were used: β-catenin CTD , γ-catenin  β-catenin NTD . Secondary Abs used were: Alexa Fluor 488 Donkey Anti-Mouse, , Alexa Fluor 488 Donkey Anti-Rabbit . Images were visualized under a fluorescence microscope  and acquired with CCD camera (Zeiss Axiocam HR).Cells were lysed in RIPA buffer containing protease inhibitor and phosphatase inhibitor tablets . Cell lysates were cleared by centrifugation and protein concentration determined by Bio-Rad Protein Assay kit . 5–30 µg (as indicated in the figure legends) of total protein in SDS sample buffer was loaded per lane and separated on NuPAGE Tris-Acetate precast polyacrylamide gels (Life Technologies). Detection of proteins was done with the following primary Abs and dilutions: β-catenin CTD , γ-catenin  β-catenin NTD , E-cadherin , α-Catenin , p120-catenin , Actin . Secondary Abs were: donkey anti-mouse IgG-HRP , donkey anti-rabbit IgG-HRP . Signals were developed using ECL Prime Western Blotting Detection Reagent  and quantification of signal intensities was done using the Image Studio Lite software .CTNNB1 exon 3 was performed by standard PCR using Phusion DNA polymerase  with primers Ex3-F (5′-caa tgg gtc ata tca cag att ctt-3′) and Ex3-R (5′-tca aaa ctg cat tct gac ttt ca-3′). For RFLP analysis the PCR products from CTNNB1 exon 3 were purified  and digested with Hpy188I restriction enzyme (New England Biolabs) before separation on 4% agarose gel .Genomic DNA was isolated using GenElute Mammalian Genomic DNA Purification Kit (Sigma-Aldrich) and PCR amplification of CTNNB1 exon 3 were TOPO cloned  and plasmid DNA was isolated  and sent for sequencing  using the M13 forward primer (5′- tgt aaa acg acg gcc agt-3′). For each of the β-catenin deficient cell clones , plasmid DNA from 10 individual bacterial colonies were sequenced.For sequencing, PCR products form CTNNB1 exon integrity in the β-catenin deficient clone #111  following the manufacturer's instructions. cDNA was synthesized using the SuperScript VILO kit (Life Technologies), and qRT-PCR was carried out using TaqMan gene expression master mix (Life Technologies) according to the manufacturer's instructions on a StepOnePlus cycler (Life Technologies). GAPDH was used to normalize the amount of cDNA in each sample and to guarantee the comparability of the calculated mRNA expression in all samples analyzed. The following TaqMan probes were used: GAPDH (Hs02758991_g1), CTNNB1 (Hs00355049_m1), JUP (plakoglobin) (Hs00158408_m1) all from Life Technologies.Single cell suspensions were fixed overnight at −20°C in 70% EtOH before resuspension in propidium iodide (PI) staining solution  for 30 minutes at RT. Flow cytometric analysis was performed on a FacsARIA (BD Biosciences) where the pulse width versus pulse area signal was used to exclude doublets. For each sample 50 000 cells were acquired.http://www.doubling-time.com/compute.php (equation used: amount  = 0.5288×e0.0317×time).For measurement of cell proliferation the IncuCyte FLR live cell imaging system  was used to quantify the confluency of living cells. Calculation of the doubling times (Td) was done using the online calculator at 2 (Sigma-Aldrich). 48 hours after transfection the cells were harvested in passive lysis buffer and both Firefly and Renilla luciferase activity was measured in the same sample using the Dual-Luciferase Reporter Assay System (Promega). The luminescence was quantified on a GloMax-Multi microplate reader (Promega) according to the manufactures instructions.The day before transfection 200 000 cells were plated out in 12 well dishes. For each transfection, 1.8 µg of the TCF/LEF reporter pSuperTopFlash (8xTCF/LEF binding sites driving Firefly luciferase expression) CTNNB1 gene disrupted clones  in Light and Heavy media, respectively the cells were harvested and lysates from each of the CTNNB1 gene disrupted clones were combined in a 1∶1 ratio (protein concentration) with lysates from wild type cells. After enzymatic digestion the samples were subjected to liquid chromatography-tandem mass spectrometry and isotopic peptide pairs were quantified with proteome discoverer . Perseus (www.maxquant.org) was used to generate the heat map and Ontology enrichment analysis was done using DAVID Detailed description of the SILAC analysis is included in the CTNNB1 gene disrupted clones #4 and #111. The RNA was subjected to microarray analysis using Illumina HumanHT-12 v4 Expression BeadChips  at the Norwegian Genomics Consortium core facility . For each sample 6 biological replicates were analyzed. Data extraction and quality control was performed in GenomeStudio (Illumina) and the data analysis was performed using J-Express Total RNA  was isolated from wild type BxPC-3 cells and 5–1×106) were resuspended in 500 µl Annexin V binding buffer before addition of 1 µl of 5xAnnexin V-FITC apoptosis detection reagent . After 5 minutes incubation at RT the number of FITC positive cells were quantified by flow cytometry .Cells  as negative control. Dynabeads/antibody complex was incubated with 400 µg protein on a rotation wheel at 4°C over night. Immunoprecipitated proteins were eluted for 5 min at RT on a spinning wheel with 2×40 µl elution buffer (0.1 M citric acid). 20 µl 5x loading buffer (LB) for SDS-PAGE was added to the eluates, and 30 µl was further resolved by NuPAGE Novex 3–8% Tris-Acetate (Life Technologies) together with 10 µg input (positive control), immunoblotted and probed with primary antibodies against mouse γ-catenin  and mouse E-Cadherin . Primary antibodies were visualized with HRP-conjugated antibodies (Santa Cruz Biotechnology) (1∶10000) and chemiluminescent substrate ECL Prime Western Blotting Detection Reagent .Cultured cells were trypsinized and lysed for 30 min on ice in cold non-denaturing lysis buffer  containing protease inhibitor cocktail . Lysates were spun for 15 min at 13 000 rpm to remove cell debris, and protein concentrations were measured using Quick Start Bradford 1x Dye Reagent (Bio-Rad Laboratories) according to the manufacturer's instructions. 50 µl Dynabeads Protein G magnetic beads (Invitrogen) was used according to the manufacturer's instructions. 5 µg anti-rabbit plakoglobin (γ-catenin) antibody  was bound to pre-washed beads in 100 µl MQ HThe day before transfection 200 000 cells were plated out in 6 well plates. Transfection were done with 40 nM of either MISSION siRNA Universal Negative Control #1 with no homology to all mature and predicted RefSeq mRNA sequences  or MISSION plakoglobin siRNA  using 5 µl of LF2000 in a total of 2.5 ml growth medium without antibiotics. After 24 hours the medium was replaced and the cells were incubated for another 48 hours before analysis/harvesting.Error bars in figures indicates standard deviation (SD) of the mean from three measurements in an experiment. Student t-test (unpaired) was performed to access statistical significance between the mean CTNNB1 relative quantification (RQ) value of three parallel measurement from BxPC3ΔCTNNB1 clone #4 versus clone #31 and clone #79 versus clone #31 as indicated in Although functional studies based on the convenient knockdown strategy by siRNA can provide significant information on the function of a targeted protein, siRNA only reduces the amount of a given protein, leaving the possibility that the protein can maintain some of its core functions CTNNB1)  . A knockAfter transfection of the PA cell lines with mRNA of β-catenin exon 3 specific ZFNs , clones CTNNB1 gene (incorporating the ZFNs binding sites), was carried out  with primers spanning exon 3 of the ried out . In clonried out . To testried out . As experied out . In concried out . To get CTNNB1 gene in exon 3 resulting in a disruption of the β-catenin reading frame leading to a loss of protein expression.In summary, by employing the ZFN technology, we have isolated 5 individual clones of BxPC-3 cells that contained targeted deletions of the CTNNB1 gene that would disrupt the reading frame  recognizing the C-terminal domain (CTD), no β-catenin staining could be observed in the isolated clones  using exposure settings that generated an optimal β-catenin signal in wild type cells  PCR was carried out on the wild type and β-catenin deficient clones. Interestingly, in addition to a loss of β-catenin protein , the cloA levels . Clone #2 to block β-catenin degradation and thereby stimulate WNT signaling, a 25 fold increase in the STF reporter activity was observed (compared to the control SFF activity) demonstrating the presence of a viable WNT/β-catenin signaling pathway at the level of the destruction complex and downstream in BxPC-3 cells. In contrast, all β-catenin deficient clones neither showed a STF basal activity nor the ability to induce STF activity upon GSK3β inhibition, indicating that β-catenin mediated transcriptional activation by TCF/LEF in this cellular model cannot be rescued by alternative proteins or pathways.One of the main functions of β-catenin is to mediate canonical WNT signaling through association with transcription factors predominantly of the TCF/LEF family to activate target gene expression To examine whether the depletion of β-catenin affected cell growth, cells were grown in an IncuCyte live cell imaging system. As seen in To test whether mutant BxPC-3 clones showed altered levels of apoptosis, an AnnexinV assay was carried out. As seen in Overall, these data suggest that β-catenin is not essential for normal survival in BxPC-3 cells although its depletion can lead context dependent to reduced proliferation rates.CTNNB1 gene disrupted clones β-catenin protein was detected. This was most likely due to exclusion from the list of quantified proteins, since for proteins that are absent or at very low levels in either of the heavy or light labeled cell lysates, a ratio of peak intensities will not be generated in the SILAC analysis and the protein will thus be excluded. To identify relevant shared biological functions associated with the up and down regulated proteins (<0.67 or>1.5 fold change), Gene Ontology (GO) term enrichment analysis was performed on those differentially expressed proteins that were quantified in at least three of the five CTNNB1 gene disrupted clones. For the 45 up regulated proteins eight significantly enriched GO terms were identified while for the 100 down regulated proteins 16 significantly enriched GO terms were identified  was performed entified .In the SILAC analysis, the proteins identified to be associated with cell adhesion included components of integrins , laminins (LAMB1), focal adhesion , tight junctions (F11R) and desmosomes (DSG2 and PKP1) . MoreoveCTNNB1 gene disrupted clones (#4 and #111) identified 85 transcripts that were the most differentially regulated  with a shift towards expression of both the long and the short p120-catenin isoforms were the same clones that displayed reduced proliferation rates .Given that plakoglobin complemented for the loss of β-catenin in adherens junctions , we nextTo further characterize the underlying mechanisms for the observed morphological changes in the BxPC-3 cells lacking both β-catenin and plakoglobin the localization and the amount of the key adherens junction proteins α-catenin, p120-catenin and E-cadherin was examined by immunostaining and WB analysis respectively. Immunostaining of p120-catenin revealed that following treatment of the β-catenin deficient clones with plakoglobin siRNA the localization of p120-catenin was changed from predominantly membranous to cytoplasmic .When the localization and protein levels of α-catenin was analyzed in cells deficient for both β-catenin and plakoglobin, the result was similar to the analysis of p120-catenin, with loss of membranous localization and reduced protein levels in some sub-clones . Also thIn summary, the combined loss of β-catenin and plakoglobin in BxPC-3 cells was associated with a reduction of p120 and α-catenin together with an internalization of E-cadherin leading to an impaired ability to form normal cell to cell contact, but not affecting overall cell growth under the tested conditions.CTNNB1 gene (β-catenin) coding sequence caused by introduction of site specific genomic DSBs. In all three cell lines that were treated with ZFNs the formation of genomic DSBs as mutations in CTNNB1 exon 3 could be observed when gDNA from the pool of transfected cells was analyzed 2 days after transfection (not shown), but only form the BxPC-3 cell line viable, β-catenin deficient clones, were obtained. The failure of isolating β-catenin deficient clones in the PANC-03.27 and PANC-1 cell lines could be due to aneuploidy or due to a reliance of the PANC-03.27 and PANC-1 cell lines on functional WNT signaling for cell proliferation and survival. Interestingly, both PANC-1 and BxPC-3 cell lines exhibit low levels of baseline WNT/β-catenin transcriptional activation KRAS  contained small deletions (1-31 bp) exclusively in the on-target region between the binding sites for ZFN-1 and ZFN-2  was found to be down regulated  while inAdherens junctions are dynamic structures with the ability to continuously being formed and disassembled whereby the main mechanism controlling adherens junction homeostasis is the regulation of the amount of E-cadherin present at the cell membrane The data presented in this work help to identify core functions of β-catenin in PA cells, and highlight compensatory mechanisms that the tumor cells can activate to attenuate the effect of a β-catenin depletion. This has implications for designing therapeutic strategies based on selective pathway inhibitors.S1 FigImmunostaining  of plakoglobin siRNA treated WT and CTNNB1 gene disrupted clones.(PDF)Click here for additional data file.S1 TableGene Ontology enrichment analysis of proteins quantified by SILAC to be up or down regulated in at least three of the β-catenin deficient clones.(PDF)Click here for additional data file.S2 TableList of the most differentially regulated probes from significance analysis of microarrays (SAM) comparing wild type BxPC3 cells and the gene disrupted clones #4 and #111 (average).(PDF)Click here for additional data file.S3 TableGene Ontology enrichment analysis of the most differentially regulated transcripts between the wild type BxPC3 cells and the β-catenin deficient clones #4 and #111 (average).(PDF)Click here for additional data file.S4 TableComplete annotated heat map generated from the proteins identified and quantified by SILAC in at least three β-catenin deficient clones.(PDF)Click here for additional data file.S5 TableList of all proteins detected and quantified by SILAC in the individual β-catenin deficient clones.(XLSX)Click here for additional data file.S1 Materials and MethodsQuantitative proteonomics by SILAC.(DOCX)Click here for additional data file.


Here we examined how TASK channels are involved in IR-dependent activation/recruitment of MNs in the rat dl-TMN by using multiple methods. The real-time PCR study revealed that single large MNs (>35 μm) expressed TASK1 and TASK3 mRNAs more abundantly compared with single small MNs (15–20 μm). The immunohistochemistry revealed that TASK1 and TASK3 channels were complementarily distributed in somata and dendrites of MNs, respectively. The density of TASK1 channels seemed to increase with a decrease in soma diameter while there were inverse relationships between the soma size of MNs and IR, resting membrane potential, or spike threshold. Dual whole-cell recordings obtained from smaller and larger MNs revealed that the recruitment of MNs depends on their IRs in response to repetitive stimulation of the presumed Ia afferents. 8-Bromoguanosine-cGMP decreased IRs in small MNs, while it hardly changed those in large MNs, and subsequently decreased the difference in spike-onset latency between the smaller and larger MNs, causing a synchronous activation of MNs. These results suggest that TASK channels play critical roles in rank-ordered recruitment of MNs in the dl-TMN.Because a rank-ordered recruitment of motor units occurs during isometric contraction of jaw-closing muscles, jaw-closing motoneurons (MNs) may be recruited in a manner dependent on their soma sizes or input resistances (IRs). In the dorsolateral part of the trigeminal motor nucleus (dl-TMN) in rats, MNs abundantly express TWIK (two-pore domain weak inwardly rectifying K channel)-related acid-sensitive-K The principle of the rank-ordered recruitment was originally proposed based on the conduction velocity of the motor nerve fibers innervating a set of muscle fibers. After a half-century, we for the first time disclosed the molecular mechanism for the rank-ordered recruitment of jaw-closing MNs.The mastication of foods occurs during the slow-closing phase of the mastication cycle, in which the isometric contraction of jaw-closing muscles is developed through the rank-ordered recruitment of jaw-closing motoneurons (MNs). However, its molecular mechanism remains unknown. Here we show that the TWIK (two-pore domain weak inwardly rectifying K channel)-related acid-sensitive-K+ channel (TASK)-1 and TASK3 channels are the major determinants of the resting membrane potential and input resistance -related acid-sensitive-Kance IR; . TASK1 aance IR; . These cance IR; . The sinance IR; .Motoneurons (MNs) in the dorsolateral part of the trigeminal motor nucleus dl-TMN;  highly e+ currents in basal forebrain cholinergic neurons are activated by nitric oxide (NO) donor and 8-bromoguanosine-cGMP  were anesthetized with chloral hydrate  and transcardially perfused with PBS. The brain was quickly removed from the skull and immediately flash frozen with dry ice. Serial cryostat sections (20 µm) of the dl-TMN were cut and mounted on polyethylene terephthalate (PET) membrane slides (Leica Microsystems). Sections were air dried at room temperature for 30 s, fixed with 70% ethanol for 1 min, washed in RNase-free diethylpyrocarbonate (DEPC)-treated water and stained with 0.05% toluidine blue for 1 min Aa. AfterPET slides were mounted on a Leica AS LMD System (Leica Microsystems) with the section facing downward. Dye-labeled MNs were selected for capture and divided into two groups according to their mean diameters. The neuron whose mean diameter was 15–20 µm was categorized as a small MN Ab, whilem each primer and 2 µl of reverse transcription product, through the following series of reactions: 95°C for 20 s, followed by 40 cycles , with a Step One Real-Time PCR System (Applied Biosystems). The following primers were used for PCR: rat TASK-1: forward, 5'-CGGCTTCCGCAACGTCTAT-3', and reverse, 5'-TTGTACCAGAGGCACGAGCA-3'; rat TASK-3: forward, 5'-GACGTGCTGAGGAACACCTACTT-3', and reverse, 5'-GTGTGCATTCCAGGAGGGA-3'; and GAPDH: forward, 5'-GAGAATGGGAAGCTGGTCATCAAC-3' and reverse, 5'-ACTCCACGACATACTCAGCACCAG-3'. Standard curves were generated for each set of primers using serial dilutions of rat brain cDNA to ensure a similar efficiency of amplification . All the primers were obtained from Sigma Genosys. Aliquots of the PCR products were analyzed by 2% agarose gel electrophoresis  and were visualized using ethidium bromide. The PCR product was run in parallel with known molecular weight markers . The amplified PCR fragments obtained from large MNs were detected in accordance with the respective lengths predicted by the primers (T) method  according to the manufacturer instructions, during which samples were subjected to DNase digestion using DNase I (Qiagen) to remove any contaminating genomic DNA. RNA was then reverse transcribed for 1 h at 37°C using Sensicript Reverse Transcription kit (Qiagen). Real-time PCR was performed in a reaction mixture (20 µl) composed of 10 µl of 2× Fast SYBR Green Master Mix (Applied Biosystems), 0.35 m primers B. The da) method , where tS) and a certain number of large MNs (NL) are proportional to NS × VS and NL × VL, respectively, given that VS and VL are the mean cell volumes of the sampled small and large MNs, respectively. When NS × VS is equal to NL × VL, VS/VL is equal to NL/NS. The normalized GAPDH mRNA in a single small MN compared with that in a single large MN is proportional to NL/NS. Therefore, we evaluated the relationship between the cell number and CT of GAPDH mRNA obtained from 50, 200, and 400 small MNs  and that between the cell number and CT of GAPDH mRNA obtained from 10, 50, and 80 large MNs . CT represents the fractional cycle number at which the baseline-corrected normalized fluorescence signal (ΔRn) reaches a threshold value (0.03).Because the housekeeping gene GAPDH mRNA level is likely to be proportional to the cell volume, the GAPDH mRNA levels obtained from a certain number of small MNs  of rat TASK1  was syntm phosphate-NaOH buffer at pH 7.2. The cells were stored at 4°C in PBS containing 0.02% sodium azide. The cells were incubated overnight with 1 μg/ml affinity-purified guinea pig anti-rat TASK1 antibody in PBS containing 0.3% Triton X-100, 0.12% λ-carrageenan, 0.02% sodium azide, and 1% normal donkey serum (PBS-XCD); and then were incubated for 1 h with 10 μg/ml biotinylated donkey anti-guinea pig IgG antibody  in PBS-XCD. Subsequently, the cells were incubated for 1 h with avidin–biotin enzyme complex  in PBS containing 0.3% Triton X-100 (PBS-X). Furthermore, the sections were incubated for 30 min with biotinylated tyramine (BT)-glucose oxidase (GO) reaction mixture containing 1.25 μm BT, 3 μg/ml GO , 2 mg/ml β-d-glucose, and 1% BSA in 0.1 m PB, pH 7.4. After a rinse with PBS-X, the sections were incubated for 1 h with 5 μg/ml Alexa Fluor 594-conjugated streptavidin  to stain TASK1 and then were incubated for 5 min with DAPI to stain nuclei.The specificity of anti-TASK1 antibody was examined using HEK293 cells transfected with TASK1 or mock. For the transient expression of TASK1, HEK293 cells  cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin were transfected with pIRES2-ZsGreen1-TASK1 by using Lipofectamine LTX (Invitrogen) or the empty vector pIRES2-ZsGreen1 mock; . For immFor immunostaining, fixed brains were prepared from three Wistar rats following the protocol described in the previous study . The 40-The procedure for slice preparation was the same as that in the previous study . Using Wm): 124 NaCl, 1.8 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.2 KH2PO4, and 10 glucose, bubbled with a mixture of 95% O2/5% CO2. The internal solution had the following composition (in mm): 123 K-gluconate, 8 KCl, 20 NaCl, 0.5 MgCl2, 2 ATP-Na2, 0.3 GTP-Na3, 10 HEPES, and 0.1 EGTA; the pH was adjusted to 7.3 with KOH. In some experiments, 123 mm K-gluconate, 8 mm KCl, and 20 mm NaCl were substituted for 118 mm K-gluconate, 18 mm KCl, 14 mm NaCl, and 10 mm biocytin to characterize the morphology of MNs. The patch pipettes had a DC resistance of 4–5 MΩ when filled with the internal solution. The membrane potential values given in the text were corrected for the junction potential between the internal solution (negative) and the standard extracellular solution (10 mV). All recordings were made at room temperature (20–24°C). The sealing resistance was usually >10 GΩ. Whole-cell currents or voltages were low-pass filtered at 2 kHz (four-pole Bessel filter) and digitized at a sampling rate of 10 kHz . Under the current-clamp condition, depolarizing and hyperpolarizing current pulses were applied every 30 s. The IR was calculated from the I–V relationships, which were usually linear when measured at the peak timing of the hyperpolarizing responses or sag potentials evoked immediately after the onset of negative current pulses  and injection with cRNA were performed as described previously (m): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4 with NaOH, and supplemented with gentamicin (50 μg/ml). Currents were recorded 1–3 d after the cRNA injection.The isolation and maintenance of the oocytes of frogs (eviously . Mouse Teviously  and pIREm): 90 KCl, 3 MgCl2, 0.15 niflumic acid, and 5 HEPES, pH 7.4 with KOH. The tip resistance of the glass electrodes was 0.4–1.5 MΩ when filled with the 3 m KCl pipette solution. Niflumic acid  was added in the bath solution to block Ca2+-dependent Cl− channels, which are richly expressed in the plasma membrane of oocytes (m. The currents were measured 5–10 ms before the offset of the voltage step.Membrane currents of oocytes were recorded by two-electrode voltage clamp using the GeneClamp 500 amplifier (MDS) at room temperature. Data were reproduced and analyzed with pCLAMP version 10 (MDS) and Clampfit version 10.2 (MDS). The bath solution contained the following (in m oocytes . 8-Br-cGm N-(3-triethylammoniumpropyl)-4-(4-(4- (diethylamino) phenyl) butadienyl) pyridinium dibromide , which was attached to a microscope  equipped with a water-immersion objective . The imaged area was 1.6 × 1.6 mm2, and each pixel (element) of the 80 × 80 array detected the optical signals generated by a square region (20 × 20 μm2). The fluorescence images were captured at a sampling rate of 1000 Hz. Repetitive electrical stimulation (10 pulses at 100 Hz) of 100 μs duration was delivered via a sharp monopolar tungsten electrode  placed just dorsal to or in the dorsal part of the TMN to simulate presumed Ia afferents (m. The amplitudes of the 1st to 9th (before 8-Br-cGMP application) or the 10th (after 8-Br-cGMP application) optical responses at the region of interest placed in the dl-TMN . A p value <0.05 was considered statistically significant.Statistical analysis was performed using STATISTICA10J (StatSoft). Numerical data were expressed as the mean ± SD. The statistical significance was assessed using ANOVA followed by Fisher’s PLSD p < 0.001, paired t test; n = 7 samples), while the former was 20 times higher than the latter in small MNs  and large (>35 μm) MNs in the dl-TMN using a real-time PCR method. The representative example of quantitative PCR for TASK1, TASK3, and GAPDH mRNAs obtained from 80 large MNs and that obtained from 400 small MNs are shown in T of GAPDH mRNA  of large spheres to small ones (diameter ratio >35/20), the volume ratio of large polygonal neurons to small ones can be smaller, depending on their shapes (35/20 > axis ratio > [35 × cos (θ1)]/[20 × cos (θ2)], when θ1 is larger than θ2; 0 < θ < 90˚), given that polygonal shape is a quadrangular pyramid. Based on the assumption that GAPDH content in a single large MN is five times larger than that in a single small MN, we estimated the expression levels of TASK1 and TASK3 mRNAs in single large and small MNs. The expression level of TASK1 mRNA in one large MN was estimated to be 2.1 times higher than that in one small MN , while that of TASK3 mRNA in one large MN was estimated to be 20 times higher than that in one small MN , we first examined its selectivity using HEK293 cells transfected with TASK1 or mock together with ZsGreen1. In the HEK293 cells represented by DAPI staining Ab,Bb, TAin situ hybridization in previous studies  than that obtained from larger MNs  than that obtained from larger MNs . An inwardly rectifying current (Iir) became apparent at potentials more negative than −100 mV as represented by an inwardly rectifying I–V relationship measured just before the offset of current pulses . This observation clearly indicates that 8-Br-cGMP decreased the IR through an enhancement of leak K+ currents without causing contamination by other conductances. In contrast, the inwardly rectifying component was not markedly affected by 8-Br-cGMP  between the two parameters . Taken together, these results suggest that 8-Br-cGMP decreases IRs in smaller MNs in the dl-TMN due to the activation of TASK1 channels, while it either hardly changes or slightly increases those in larger MNs in the dl-TMN, presumably due to the opposing effects of 8-Br-cGMP on TASK1 and TASK3 channels  different between the smaller and larger MNs.In the four pair recordings obtained from two MNs of different sizes that were visually identified under Nomarski optics, the IR and the latency to the first spike were measured before and after the application of 8-Br-cGMP and were plotted in the two-dimensional space defined by the IR and the latency to the first spike C. The smp = 0.032, Wilks’ lambda) changed the distribution of the IR and the latency to the first spike in the two-dimensional space in smaller MNs  increased from 31.3 ± 2.9 to 39.5 ± 3.1 ms following the application of 8-Br-cGMP  difference in the mean value of the IRs obtained before and after the application of 8-Br-cGMP . In contrast, in larger MNs, 8-Br-cGMP did not significantly  change the distribution of the IR and the latency to the first spike  or in the mean value of the latencies to the first spikes  obtained before  and after  the application of 8-Br-cGMP.The discrimination analysis revealed that 8-Br-cGMP significantly  different from those in larger MNs  difference in the distribution of the IR and the latency to the first spike between the smaller and larger MNs. Subsequently, the mean value of the difference in the latency to the first spike between the smaller and larger MNs was significantly  decreased from 18.0 ± 4.2 to 6.3 ± 8.9 ms  and at pH 8.4 . As it has previously been reported , but not at pH 8.4 . Based on these results, it can be concluded that 8-Br-cGMP enhances TASK1 currents, whereas it suppresses TASK3 currents.TASK3 currents were evoked by voltage steps ranging between −150 and +60 mV by 30 mV steps applied at a holding potential of −90 mV. When the extracellular pH was decreased from 8.4 to 7.4, TASK3 currents evoked in response to positive voltage steps ranging between 0 and +60 mV were markedly decreased A,D. Whenecreased D. The poreported  that 8-Bm 8-Br-cGMP, the excitation spread in response to the first stimulation appeared to be decreased compared with the control responses  compared with that obtained before the application of 8-Br-cGMP (0.73 ± 0.08), while that induced by the 10th stimulation after the application of 8-Br-cGMP (1.13 ± 0.04) was significantly larger  compared with that obtained before the application of 8-Br-cGMP . Thus, 8-Br-cGMP suppressed early optical responses but facilitated late optical responses, indicating the inhibition of the earlier recruitment of smaller MNs and the facilitation of later recruitment of larger MNs, respectively, causing a more synchronous activation of small and large MNs in the dl-TMN. This is consistent with the effects of 8-Br-cGMP on the IRs of smaller and larger MNs and on EPSPs  applied just dorsal to or in the dorsal part of the TMN A are modesponses D, 8-Br-c-cGMP 1; E, n = 5.on EPSPs , and alson EPSPs . Thus, T+ channels is invariable irrespective of cell sizes.The immunohistochemistry revealed that TASK1 and TASK3 channels were complementarily distributed in soma and dendrites of MNs, respectively , suggestin situ hybridization demonstrated that the expression level of TASK3 mRNA in MNs appeared to be higher than that of TASK1 mRNA. This discrepancy can be explained by the underestimation of TASK3 mRNA levels in the present study because dendrites expressing TASK3 had been cut by LMD . The expression level of TASK1 channels in one large αMN is 2.1 times higher than that in one small αMN, provided that there was no difference in the expression level of TASK1 mRNA between γMNs and small αMNs F. HoweveDuring the slow-closing phase of the mastication cycle , the iso+ currents in hypoglossal MNs are mediated by the homodimeric TASK3 and heterodimeric TASK1/3 channels, respectively . This suggests that the expression level of TASK3 channels might have been much higher than that of TASK1 channels in Xenopus oocytes. Provided that PKG activation by 8-Br-cGMP is not enough to phosphorylate all the TASK3 channel substrates in single Xenopus oocytes, the effects of 8-Br-cGMP on TASK3 currents would be small. Thus, it is not reasonable to directly compare the effects of 8-Br-cGMP on TASK1 currents with that on TASK3 currents in Xenopus oocytes. Therefore, the modest effect of 8-Br-cGMP on TASK3 channels compared with TASK1 channels is not necessarily contradictory to the negligible effect of 8-Br-cGMP on the IRs in large αMNs. These results suggest that 8-Br-cGMP decreases the time necessary for recruitment starting with smaller αMNs and progressing to larger αMNs (In small αMNs, 8-Br-cGMP hyperpolarized the resting membrane potentials and decreased the IRs A, 7A, anger αMNs C. Consisger αMNs .+ currents without marked effects on the Iir (Together, given NO release in the TMN from nitrergic neurons during the isometric contraction of JC muscles A, the sp the Iir Ac, it is


In addition, in the superconducting phase, the shape of the splitting in k space is modified by correlations.We carry out a theoretical study of the bilayer single-band Hubbard model in the undoped and in the superconducting phases by means of the variational cluster approach. In particular, we focus on the splitting between the “bonding” and “antibonding” bands induced by the interlayer hopping, as well as its interplay with strong correlation effects. We find that the splitting is considerably suppressed in both the normal and superconducting phases, in qualitative agreement with experiments on Bi Tc superconductors (HTSC) takes place in the two-dimensional CuO2-layers. On the other hand, several classes of HTSC exist with a different number of CuO2-layers per unit cell, their transition temperature being strongly related to this number  in the two-dimensional Brillouin zone. The spectrum shows the asymmetric behavior of electron and hole filling induced by t′: electrons are expected to first enter the Brillouin zone around , while holes first enter at . The interlayer hopping introduces a splitting between the kz=0 and kz=π spectra, which for simplicity of language we will refer to as the bonding and antibonding bands [kx=ky the two Fermi points for the bonding and antibonding bands are exactly one over the other. When going away from this diagonal, the splitting grows until reaching a maximum near the  and  points. In Fig. π), which clearly shows the interlayer splitting. The splitting is approximately ΔU=0.32t, which is reduced with respect to the value Δ0=0.4t in the noninteracting case. The spectral function ng bands . Withoutter at π/,π/2. Theπ,0) in the superconducting case are displayed in Fig. At optimal doping, no bilayer splitting could be resolved in ARPES measurements of BSCO-2212 . In ordeπ,0) region of the optimally doped monolayer (BSCO-2201) and bilayer material (BSCO-2212) are similar [Moreover, it was found that the shape of the quasiparticle peak in the . In plitting . We check-dependence in the overdoped region. Our results suggest a reduction of the splitting at  by about 30 % in the normal and by about 70 % in the superconducting phase with respect to the tight-binding model. Moreover, in the superconducting phase, also the shape of the k dependence is modified. This larger suppression in the superconducting phase is in qualitative agreement with experiments [U=0 by introducing “by hand” a superconducting symmetry breaking field equal to the one obtained variationally at U=8. As one can see from the figure, the superconducting gap only produces a small (about 10 %) reduction, which is uniform in k. The anomalous behavior of Fig. In Fig. eriments . In ordeU=8 plotted in Fig. kz defining a bonding and antibonding band. The k dependent splitting is defined as the distance between the maxima of these peaks for kz=0,π. For the superconducting state, we determine the splitting for the quasiparticle states below the Fermi level. We have checked that it very close to the splitting of the mirror states above it. When going away from the antinodal point both in the normal state as well as in the superconducting state, each quasiparticle peak first broadens, which introduces an error in the determination of Δ, and then evolves into a two peak structure, which resembles the peak-dip-hump structure that is observed in ARPES [The values of the splitting for in ARPES . MeasuriWe have studied the bilayer Hubbard model by means of the variational cluster approach, a method appropriate to capture short range correlation in strongly interacting lattice systems. As expected, the interlayer hopping splits the spectrum into a bonding and an antibonding band. However, the corresponding bilayer splitting is strongly renormalized due to correlations. This is evident in the overdoped case in both the normal and superconducting phases. In qualitative agreement with ARPES measurements, the suppression effect is stronger in the superconducting phase. Surprisingly, for optimal doping, the bilayer splitting vanishes completely, as found in ARPES .


In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation.Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium.  Cell proliferation and differentiation are highly regulated and ordered processes in the intestinal tract which depend on a complex array of signaling molecules that provide the gut with the ability to respond to a wide range of physiological and pathophysiological perturbations. When activated inappropriately, the same signals can cause or contribute to malignant transformation and the development of colorectal cancers. Wnts, for instance, which comprise a large family of evolutionarily conserved cysteine-rich stem cell growth factors, are differentially expressed throughout the gut and provide the necessary cues for normal epithelial cell development112Enteric microbes also play a role in regulating host functions and gene expression of the gut epithelium910121314316In this study, we report the interaction between pericrypt-derived myofibroblast-derived Wnt-5a and Hsp25/27 in the regulation of colonic epithelial proliferation. We find that pericrypt myofibroblast-derived Wnt-5a is proteolytically converted to a soluble bioactive, 36 mer peptide that likely facilitates its delivery to epithelial Frizzled receptors in the lower part of the colonic crypt to promote cell proliferation and development, an effect mediated through the canonical activation of β-catenin. However this action alone is insufficient and requires simultaneous inhibition of butyrate-induced Hsp25/27 in cells in the proliferative compartment of colonic crypts. As Wnt-5a signaling declines in the upper crypt that lies outside of the pericrypt myofibroblast sheath, butyrate-induced Hsp25/27 expression becomes more prominent as a result of diminishing negative regulation by Wnt-5a.in vivo effects of Hsp25 expression on crypt cell proliferation can be seen in a segment of proximal colonic mucosa exposed to luminally-administered Hsp25 transgene-containing lentivirus. Normally, Hsp25 expression is confined to the upper third of colonic crypts−/− mice, robust Hsp25 protein expression was observed by immunostaining throughout the colonic crypt epithelium The short chain fatty acid (SCFA), butyrate (5 mM), inhibits proliferation of murine colonic epithelial YAMC cells measured by the WST-1 assay . These dithelium , associaithelium . On the Wnts are among many myofibroblast-derived factors that regulate epithelial cell proliferation and development2in situ hybridizationThe profile of Wnt gene expression was next assessed by PCR . Among t++ dependent pathways. CM from VUPF myofibroblasts treated with Wnt-5a siRNA failed to block the anti-proliferative actions of butyrate measured by 3H-thymidine incorporation . This region of Wnt-5a is highly conserved among Wnts and its formation could be a result of enzymatic cleavage by serum or cellular tryptase .The Wnt-5a gene encodes a protein of approximately 40 kDa molecular weightTo test the functional properties of this peptide, the Wnt-5a peptide region from aa241 to aa271 (GVSGSCSLKTCWLQLADFRKVGDALKEKYDS) was synthesized and purified by HPLC (purity 92.7%). This shorter peptide was made because of the difficulty of preparing and purifying the full length 36 mer endogenous Wnt-5a peptide by the commercial vendor. As shown in waf-1 and p27. Both proteins bind to and inhibit the activity of cyclin-CDK2 or -CDK4 complexes, and thus function as a regulator of cell cycle progression at G1. Hsp25 induction through lentiviral delivery (L25) induces expression of both p21waf-1 and p27 under basal conditions . To determine if the Wnt-5a peptide might prevent the damage or subsequent recovery that involves a wound healing response, mice were treated with DSS and then allowed to recover with or without Wnt-5a peptide. Mice not treated with the peptide did not recover from the loss of body weight 17 days after DSS removal while those treated with the peptide demonstrated full recovery of body weight . Colon lwaf-1 and p27 and the maturational process begins. Thus, the regional confinement of Wnt-5a signaling and interaction with butyrate are important in maintaining the orderly development and turnover of colonic epithelial cells 36372339Another important finding of this study is the discovery that pericrypt myofibroblast-derived Wnt-5a is proteolytically converted to a soluble bioactive peptide that likely facilitates its delivery to epithelial Frizzled receptors in the lower half of the colonic crypt to promote cell proliferation and development, an effect mediated through the canonical activation of β-catenin. Our studies therefore provide a plausible explanation for how pericrypt myofibroblast Wnt-5a and possibly other Wnts function as paracrine growth factors see . The proThe process through which Wnt-5a is converted to a smaller bioactive peptide is currently unclear, but may involve intracellular processing or conversion through extracellular tissue or serum tryptases that could emanate from many several potential sources, including epithelial, mesenchymal and mast cells  or published Wnt primersYAMC cell Hsp25 was silenced as previously described using a silencing oligonucleotide  designed using RNAi designer (Invitrogen). For VUPF myofibroblast Wnt-5a silencing, a silencing oligonucleotide  or scrambled oligonucleotide (ID#4611) was used.For FrzB or Wnt-5a adenovirus, human FrzB and mouse Wnt-5a were PCR-amplified, subcloned into pAdTrack-CMV and recombinant adenovirouses generatedLentiviral constructs were made using a human immunodeficiency virus-derived systemImmunohistochemical staining was performed on sections from formalin-fixed, paraffin-embedded mouse colon for smooth muscle actin, Hsp25, and Wnt-5a or Ki-67 using the Vectastain Elite ABC kit  or EnVision+System . Slides were counterstained with hematoxylin.in vivo, a modification of in vivo silencing was usedTo reduce Wnt-5a expression in vivo, the mouse abdominal wall was surgically opened and 1 cm section of the cecum clamped. Murine Hsp25- or green fluorescent protein-encoding lentivirus was introduced in into the mouse colon lumen in PBS and left for one hour. The segment was unclamped, returned to the abdominal cavity, and the abdominal wall closed. After 3 days, a section at the site of lentivirus introduction was removed and fixed for immunohistochemical analysis.Mouse experiments were approved by the University of Chicago IACUC Committee protocol 71851. All methods were carried out accordance with relevant guidelines for surgery for anesthesia prior to surgery, introduction of lentivirus to mice, individual housing of mice with lentivirus in biosafety facilities, and health monitoring guidelines post surgery. To induce colonic epithelial cell Hsp25 expression YAMC cells were treated with silencing RNA oligonucleotide to mouse Hsp25 using the Silentfect . When appropriate, cells were infected with Hsp25 encoding lentivirus for 24 hours. Treatment with VUPF CM or Wnt-5a synthesized peptide was initiated immediately upon switching to 37 °C. When appropriate, either synthesized peptide was incubated with anti-Wnt-5a or goat IgG for 24 hours before addition to cells. Cells were harvested using the NE-PER kit . Protein was measured in the nuclear and cytoplasmic fractions and analyzed for β-catenin by Western blots.YAMC cells were transfected with TOP or FOP FLASH reporters using LT-1 . Conditions used were as described for β-catenin translocation. Cells were harvested and luciferase activity measured according to manufacturer’s instructions .Mouse treatments were approved by the Kyoto Prefectural University Animal Care and Use Committee protocol M25-150. C57Bl6 mice were purchased from Shimizu Experimental Animals . Mice were provided 2.5% (wt/vol) DSS in drinking water from days 0–7 and regular water days 8–15. On days 8–14, mice were injected i.p. daily with 10 μg Wnt-5a peptide or saline vehicle. Body weights were measured on days 0, 4, 7, 1, 13, and 15 and mice sacrificed and colon length measured and harvested for RNA and protein.All experiments were repeated at least three times using cells from different passages. Statistical analysis was performed using ANOVA for multivariate analyses and Bonferroni corrections. Data were expressed as means ± SE.How to cite this article: Uchiyama, K. et al. Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development. Sci. Rep.6, 32094; doi: 10.1038/srep32094 (2016).


Aberrant activation of the Wnt/β-catenin pathway is frequently found in various cancers, often through mutations of downstream components. Inhibiting β-catenin signaling in tumors with downstream pathway mutations remains challenging, due to a lack of favorable targets. On the other hand, targeting upstream components of the Wnt pathway is rather straightforward. However, it is difficult to identify tumors addicted to autocrine or paracrine Wnt signaling. Discovery of the R-spondin-ZNRF3/RNF43 signaling module and its genetic alterations in cancers represents a breakthrough in this area. Membrane E3 ligase ZNRF3 and RNF43 are critical negative feedback regulators of the Wnt pathway, which function through promoting ubiquitination and degradation of Wnt receptors. R-spondin proteins (RSPO1-4) serve as natural antagonists of ZNRF3/RNF43. To maintain strong and sustained Wnt/β-catenin signaling, cancers need to overcome ZNRF3/RNF43-mediated feedback inhibition. Indeed, mutations of RNF43/ZNRF3 and recurrent translocations of RSPO2/RSPO3 have recently been identified in various cancers. Significantly, genetic alterations in RNF43/ZNRF3/RSPO2/RSPO3 have shown promise as predictive biomarkers in pre-clinical models for the efficacy of upstream Wnt inhibitors. In this review, we will discuss the biology of the R-spondin-ZNRF3/RNF43 signaling module, cancer-associated alterations of this signaling module, and their value as biomarkers to identify Wnt-addicted tumors. The evolutionarily conserved Wnt signaling pathway plays critical roles in embryonic development and adult tissue homeostasis in all multicellular animals . Wnt proC. elegans protein PLR-1, ortholog of ZNRF3 and RNF43, also regulates Wnt receptor turnover, suggesting that this function is evolutionarily conserved [Ubiquitination-mediated turnover of Wnt receptors has emerged as a critical regulatory mechanism of Wnt pathway activity as it determines the responsiveness of cells to Wnt ligands. Cell surface FZD levels are stabilized by UBPY/USP8  and USP6onserved . ConsistThe molecular mechanism by which ZNRF3 and RNF43 recognize FZD has been elucidated . DisheveR-spondin proteins RSPO1-4) are secreted proteins that potently sensitize cells to Wnt/β-catenin signaling and Wnt/PCP signaling ,10. All  are secrR-spondin proteins are important for diverse biological processes such as sex determination, vasculature formation, and development of limb, lung, hair, nail, and muscle ,28,29,30Aberrant activation of Wnt/β-catenin signaling has long been implicated in cancer . SeveralThe initial report of RNF43 mutations was in benign pancreatic tumors called intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN) . With thWith the completion of most large-scale sequencing studies of common cancer types , the tumor types listed in versus 4.8%, Identified RNF43 mutations are mostly truncating events (non-sense mutations and frame-shift mutations) and reported missense mutations spread across the gene. This is consistent with the tumor suppressor role of RNF43. There are two recurrent hotspot mutations: G659fs and R117fs. They account for 41.7%~48.0% and 8.3%~12.0% of RNF43 mutations identified in colon cancer and endometrial cancer . These tIn just one year after the initial discovery of RNF43 mutations in IPMN and MCN, recurrent RSPO2 and RSPO3 translocations were identified in 3.0% and 8.0% of colon cancer, respectively . TransloInterestingly, RSPO2/3 translocations have also been identified in other tumor types. Recurrent RSPO2 and RSPO3 fusions are found in prostate cancers and Schwannoma ,51. In aZNRF3/RNF43 represents a powerful negative feedback mechanism identified in the Wnt pathway. Activation of Wnt/β-catenin signaling induces the expression of ZNRF3 and RNF43, which in turn promote the degradation of Wnt receptors on the cell surface and shut down Wnt signaling. The strength of this negative feedback regulation is demonstrated by the finding that the knockdown of β?-catenin in a pancreatic cancer line with wild-type ZNRF3 and RNF43 strongly increases the cell surface level of FZD, most likely through down-regulation of endogenous RNF43 and ZNRF3 . TherefoMouse genetic studies have supported a critical role of the R-spondin-ZNRF3/RNF43 signaling module in cancer. Rspo2 and Rspo3 were identified as sites of integration for mouse mammary tumor virus (MMTV)-induced mammary tumors ,55,56. Rin vivo [in vitro and inhibits metastasis in vivo.As discussed previously, R-spondin enhances both Wnt/β-catenin signaling and Wnt/PCP signaling through increasing the cell surface level of FZD. Wnt/PCP signaling has long been associated with cell movement, invasion and tumor metastasis . Althougin vivo . As compin vivo . RSPO3 oOur understanding of the R-spondin-ZNRF3/RNF43 signaling module in cancer is still evolving. An alternative mechanism of the RNF43-mediated Wnt pathway inhibition has recently been proposed . In thisin vitro is not trivial. In the past, people often used overexpression of secreted Wnt inhibitor DKK1 or sFRPs to demonstrate Wnt dependency. However, using this approach to demonstrate Wnt dependency could be complicated since overexpression of DKK1 or sFRPs can cause ER stress and yield false-positive data. Porcupine is a membrane-bound O-acyltransferase that is specifically required for the palmitoylation of Wnt ligands, a necessary step in the secretion of Wnt ligands. Identification of WNT974, formerly known as LGK974, as a highly potent and selective small-molecule porcupine inhibitor has significantly improved our ability to identify Wnt-dependent tumors [Multiple upstream Wnt inhibitors have been generated, including pan-FZD antibodies , LRP6 ant tumors . We testt tumors . Althougt tumors .in vitro [It should also be noted that RNF43 mutant tumors can be Wnt-independent. PATU8988S and PATU8988T are two pancreatic cancer cell lines derived from the same patient. Although both cell lines have the same RNF43 mutation, only PATU8988S is sensitive to porcupine inhibitor . Similarin vitro .Identification of RSPO2/RSPO3 translocations in cancer provides not only an attractive target for Wnt pathway inhibition using antibody approaches but also an intuitive predictive biomarker for RSPO2/RSPO3 antibodies. Since R-spondin sensitizes cells to Wnt ligands through inhibiting ZNRF3/RNF43, tumor cells harboring RSPO2/RSPO3 translocations should be sensitive to either direction inhibition of RSPO2/RSPO3 or inhibition of Wnt ligand secretion by porcupine inhibitors. Indeed, colorectal cancers with RSPO3 fusion are sensitive to RSPO3 antibody  and porcIn summary, RNF43 mutations and R-spondin translocation might serve as predicative biomarkers to identify Wnt-dependent tumors. Both RNF43 mutations and RSPO fusions are currently being used as patient enrollment criteria in the clinical trial of porcupine inhibitor WNT974 . The value of using such biomarkers for patient selection will be determined.The regulation of Wnt receptor turnover by the RSPO-ZNRF3/RNF43 signaling module has emerged as a key regulatory mechanism of Wnt signaling. Dysregulation of this signaling module is frequent in cancer. To maintain high and sustained Wnt/β-catenin signaling, cancer cells must inactivate this negative feedback mechanism, which can be achieved through the mutation of ZNRF3/RNF43 or overexpression of R-spondin. Importantly, such cancers are likely addicted to autocrine or paracrine Wnt signaling and sensitive to upstream Wnt inhibitors such as porcupine inhibitors and FZD antibodies. RNF43/ZNRF3 mutations and R-spondin translocation/overexpression can be used as biomarkers to identify Wnt-dependent tumors for administering upstream Wnt inhibitors in clinical trials.One major issue of using Wnt inhibitors is on-target toxicity due to the importance of Wnt signaling in several tissues such as the bone and the gut. Predictive biomarkers such as RNF43/ZNRF3 mutations and RSPO2/RSPO3 translocations serve to identify tumors exquisitely sensitive to Wnt inhibition. Administrating Wnt inhibitors to patients harboring RNF43/ZNRF3 mutations or RSPO2/RSPO3 translocations in their tumors may result in a wider therapeutic window and enable the successful clinical development of Wnt inhibitors. In addition, due to the functional redundancy of four R-spondin proteins, R-spondin antibodies might have a bigger therapeutic window than general Wnt pathway inhibitors such as porcupine inhibitors and pan-FZD antibodies in tumors with R-spondin translocation/overexpression. Another important consideration is combination strategy. Although Wnt inhibitors show single agent activities in preclinical models, stronger anti-tumor response is seen when Wnt inhibitors are combined with chemotherapeutic agents such as taxol ,72. Depe


Endometriosis is a chronic, estrogen-dependent disease associated with infertility and pelvic pain. Endometriosis is defined by the presence of extra-uterine endometrial tissue. It affects approximately 10% of reproductive-aged women. However, the underlying etiology, pathogenesis and pathophysiology remain to be fully elucidated. Knowledge of these factors is indispensable for the development of targeted therapies for prevention and treatment of endometriosis. Several studies, including those from our laboratory, have suggested that aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis. This is a review of the literature focused on the aberrant activation of the Wnt/β-catenin pathway in patients with endometriosis, and on how targeting the Wnt/targeting pathway may be a potentially effective approach for treating and/or preventing endometriosis. Endometriosis is a chronic, estrogen-dependent disease associated with infertility and pelvic pain. Endometriosis is defined by the presence of extra-uterine endometrial tissue. It affects approximately 10% of reproductive-aged women [Several studies, including those from our laboratory, have suggested that aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis –9. The WThis is a review of the literature focused on the aberrant activation of the Wnt/β-catenin pathway in patients with endometriosis, and on how targeting the Wnt/targeting pathway may be a potentially effective approach for treating and/or preventing endometriosis.Pathogenesis of endometriosis remains unclear. However, the implantation theory is the most widely accepted . Endometwww.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes). Treatment with PKF 115–584, a small-molecule antagonist of the Tcf/β-catenin complex, decreased the amount of total MMP-9 approximately 75% in epithelial cells and 85% in stromal cells in patients with endometriosis [Our previous study showed significantly higher total and active forms of MMP-9 in the menstrual epithelial and stromal cells of patients with endometriosis compared to those of patients without endometriosis . MMP-9 ietriosis –17. In aetriosis ,19. Our etriosis . Accordietriosis . These fFurthermore, a recent study showed that human endometrial basal glandular epithelial cells expressed nuclear SOX9, a Wnt target gene, and contained a rare subpopulation of cells with nuclear β-catenin . These fEndometriosis affects approximately 25%-50% of all women with infertility . HoweverPrevious findings, including those from our laboratory, suggested that the Wnt/beta-catenin signaling pathway might be aberrantly activated in infertile patients during the mid-secretory phase ,6,7. OurA study showed that increased E2 levels activate Wnt/β-catenin signaling to promote endometrial cell proliferation during the proliferative phase of menstrual cycle, whereas during the secretory phase, progesterone levels inhibit Wnt/β-catenin signaling, resulting in counterbalancing E2-induced proliferation and enhancing differentiation . In addiThe Wnt/beta-catenin pathway is involved in cell proliferation, migration, and invasion , which astudies that showed that endometriotic cells have invasive and metastatic phenotypes similar to metastatic carcinoma cells [In contrast, our previous study demonstrated that endometriotic epithelial cells and stromal cells were more invasive than those of matched eutopic endometrium of the same patients . These fma cells ,36. The ma cells . Levels ma cells . Both MMma cells . These fFurthermore, a recent study demonstrated that the activated TNFα–MMP-9-SRC-1 axis protects the ectopic endometrium from proinflammatory cytokinemediated apoptosis . EndometRecent studies demonstrated the presence of ectopic endometrium in the rectovaginal septum, in the Douglas pouch, in the rectum of human female fetuses ,40. TheyEndometriosis is histologically characterized by dense fibrous tissue mainly composed of collagen type I ,43. ExceDespite of its clinical importance, only a few studies have been conducted to evaluate new therapies for fibrosis in endometriosis. The cellular and molecular mechanisms underlying fibrosis in endometriosis remain to be fully elucidated.. These findings suggested the involvement of the aberrant activation of the Wnt/β-catenin pathway in the molecular and cellular mechanisms underlying fibrogenesis of endometriosis [Recent studies have demonstrated the involvement of activated Wnt/β-catenin signaling in fibrosis in several organs –50. Our Furthermore our previous study demonstrated that mRNA expression of fibrotic marker genes was significantly decreased by treatment with PKF 115–584 and CGP049090, small-molecule antagonists of the Tcf/β-catenin complex . Small-mPain is a major clinical problem in patients with endometriosis ,52–55. HStudies, including those from our laboratory, suggested that the aberrant activation of the Wnt/β-catenin pathway may be involved in the pathophysiology of endometriosis. The aberrant activation of the Wnt/β-catenin pathway in menstrual endometrium may facilitate development of endometriosis through increased cell migration and invasion.In addition, the aberrant activation of the Wnt/β-catenin pathway may facilitate growth of endometriosis through an increased invasive phenotype and resistance to apoptosis of endometriotic cells. Furthermore, the aberrant activation of the Wnt/β-catenin pathway in mid-secretory endometrium may result in the persistence of the proliferative phenotype and impaired decidualization in infertile patients with endometriosis. The findings from our laboratory also showed the involvement of the Wnt/β-catenin signaling pathway in the cellular and molecular mechanisms underlying fibrosis in endometriosis. A small-molecule antagonist of the Tcf/β-catenin complex prevented the progression of fibrosis and, more importantly, reversed established fibrosis in a xenograft model of endometriosis in immunodeficient nude mice. These findings suggested that targeting the Wnt signaling pathway may be a potentially effective approach for treating and/or preventing endometriosis Table .Table 1However, one major concern in targeting the Wnt/β-catenin pathway is the potential for side effects on stem cell maintenance and tissue homeostasis . Close a


The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP–PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways.Regular use of β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP–PKA cascade was assessed in complete bronchi and in cultured epithelial cells.Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol , a βCompared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP–PKA cascade.2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β WNT gene family includes 19 members encoding Wnts, which can activate three distinct signaling pathways. The best characterized canonical Wnt/β-catenin pathway implicated the inhibition of glycogen synthase kinase GSK-3β, resulting a cytoplasmic accumulation of β-catenin and its nuclear translocation 2+ signaling pathway is mediated by protein kinase C (PKC) and the Wnt/planar cell polarity (PCP) pathway activates the small G proteins Rho and the mitogen-activated proteins kinases (MAPK) cascade or alternatively triggers activation of the c-Jun-N-terminal kinase (JNK) leading to the transcription of target genes through the activator protein-1 (AP-1) stimulation 2+ and the Wnt/PCP signaling pathways are independent from LRP5/6 Wnt (wingless/integrated) is a large family of secreted glycoproteins with highly conserved cysteine residues involved in lung development and diseases 2-adrenoceptor agonists are the most potent known airway smooth muscle relaxants and they have been used for several decades to treat asthma and chronic obstructive pulmonary disease. However, regular use of β2-agonists alone may enhance non-specific airway responsiveness and inflammation, thereby deleteriously affecting control of chronic inflammatory airway diseases α and Gsβγ proteins stimulated by the G-coupled membrane receptors like β2-adrenoceptor may modulate the intracytosolic β-catenin β2-adrenoceptor agonists in human airways. Furthermore, we determined the effects of inhibitors or blockers of Wnt signaling on the β2-agonist-induced sensitizing effect in human isolated bronchi and we assessed the impact of prolonged exposure to β2-agonist on the mRNA expression of genes interacting with Wnt signaling pathways or β2-adrenoceptor/cAMP–PKA cascade in complete human bronchi and in primary cultures of human bronchial epithelial cells.The purpose of this work was to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure (15 hours) to βThe study and the consent procedure were approved by the local Ethics Committee . At least one day prior scheduled surgery for lung cancer, patients gave written informed consent that has been recorded in the individual's file. Bronchial tissues were surgically removed from 44 patients ; all were smokers or ex-smokers. Bronchial segments (inner diameter 2–3 mm) were collected from sites distant from the tumor. The absence of tumor infiltration was retrospectively established in all bronchi.2, 1.2 KH2PO4, 25 NaHCO3 and 11.7 glucose). For function, rings of similar length from the same bronchi were randomly distributed in paired groups. Half of the rings were placed in oxygenated Krebs-Henseleit solution, in parallel the other half was placed in oxygenated Krebs-Henseleit solution with 0.1 µM fenoterol  and both were incubated (37°C) for 15 hours as previously described 2+ pathway : wortmaninn (1 µM); 6) a selective peroxysome proliferator activated receptor-γ (PPAR-γ) agonist, which inhibits both noncanonical Wnt/JNK and canonical Wnt/β-catenin signaling Tissues were dissected and washed in oxygenated Krebs-Henseleit solution , transferrin (7.5 µg/ml), hydrocortisone (1 µM), endothelial cell growth supplement (2 µg/ml), epithelial growth factor (25 ng/ml), triiodothyronine (30 nM), L-glutamine (1 mM), penicillin/streptomycin (100 µg/ml), gentamycin (50 µg/ml), and amphotericin B (1 µg/ml). The cultures reached confluence between 4 and 10 days after plating and developed a transepithelial potential difference. Then, 200 µl of culture medium with or without 0.1 µM fenoterol was deposited on the apical side of each culture. After 15 hours, epithelial cells were lysed in TRIzol reagent, and then stored at −80°C until use.Tissues were processed within 24 hours after surgery. Bronchial epithelial cells were harvested using enzymatic isolation procedures to establish primary cultures, as previously described 2-5% CO2 and maintained at 37°C. Each preparation was connected to a force displacement transducer and isometric tension was recorded continuously for analysis . Preparations were suspended with an initial tension of 1.5 g in organ baths −10 to 10−7 M with logarithmic increments) After 15 hours of incubation with or without fenoterol, the bronchial rings were suspended on hooks in a 5-ml organ baths containing Krebs-Henseleit solution, gassed with 95% OFZD1-10; 2) genes related to Wnt proteins: WNT2, WNT3A, WNT5A, WNT7B, WNT10A, and WNT11; 3) genes related to LRP5/6 co-receptor: LRP5 and LRP6; 4) genes that interact with the cAMP–PKA cascade and the β2-adrenoceptor regulation: SLC9A3R1, PPP2R1A, EP300, CTBP1, and FRAT1; 5) two genes related to PKC, p38/JNK or Rho/MAPK signaling pathways: PKCE and GADD45A; 6) gene related to Wnt signaling pathway regulation: WISP1. The expression of transcripts of the relevant genes has been analyzed in the bronchi using a specific TaqMan Array based on predesigned reagents . In order to validate the extraction of intact cellular mRNA and standardize the quantitative data, three reference genes, hypoxanthine phosphoribosyltransferase (HPRT1), glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and β-glucuronidase (GUSB) were amplified simultaneously.The RT-qPCR experiments were performed as described previously t test for paired data and repeated measures ANOVA with Bonferroni adjustments for multiple comparisons . A value of P<.05 was considered statistically significant. Because inadequate statistical power cannot rule out related to small samples, the standardized effect size d for the difference between means was calculated to determine whether the observed effect of pretreatment co-incubated with fenoterol was small (|d|≥.20), medium (|d|≥.50), or large (|d|≥.80) according to the Cohen's conventions 50 (pD2) for potency. Emax (g) represented the maximal contraction induced by 0.1 µM ET-1 50) was derived graphically from the log concentration-effect curves and defined as the negative log of the ET-1 concentration achieving 50% of the maximal effect. Δ(−log EC50) represents the difference between −log EC50 obtained with the fenoterol-treated bronchi and −log EC50 obtained with their paired controls. The quantitative data obtained from RT-qPCR experiments was expressed as relative expression (2−ΔCt) t is the difference between the target gene Ct and the mean Ct of the reference genes.Values are presented as means ± SEM. The results were analyzed using Student's n = 44, P = 0.76). Fenoterol  did not change significantly the basal tone of human bronchi before contraction with ET-1 . Incubation of the bronchi with 0.1 µM fenoterol significantly enhanced the ET-1–induced contraction . −log EC50 values was 8.36±0.09 log units in the presence of fenoterol vs 8.27±0.10 log units for the paired–control rings [Δ(−log EC50)  =  0.08±0.09, n = 44, P = 0.37; Mean patted-dry weight of bronchi exposed to fenoterol was comparable with that of their paired controls  (The mRNA levels of the 3 housekeeping genes were unaffected by incubation with fenoterol or incubation time (not shown). At basal state (H0), whole human bronchi mainly expressed FZD6 mRNA while the WNTs expression was low, and WIF1 expression remained practically undetectable . Incubattivation . CompareGADD45A) . MoreoveGADD45A) . CompareGADD45A) .Blocking the extracellular Wnts or the Fzd receptors by 1 µM SFRP1 or 1 µM WIF1 increased the contractile response to ET-1 of human bronchi . When thInactivating the Wnt secretion by co-incubation with 1 µM IWP2 had no significant effect on the rise in maximal airway contraction to ET-1 elicited by fenoterol exposure for 15 hours in 10 human bronchi  and 2.Compared to paired controls, co-incubation with 1 µM DKK1 significantly decreased the fenoterol-induced rise in efficacy to ET-1 in human bronchi  and 2.In absence of fenoterol, 1 µM wortmaninn and 1 µM Y27632 produced opposite effects on the bronchial contraction to ET-1 . BlockagAs seen in 2-agonist with high intrinsic efficacy, sensitized human bronchi at least in part via the Wnt/β-catenin signaling, notably the LRP5/6 co-receptor and the nuclear recruitment of TCF/LEF. Conversely, fenoterol exposure did not change the Wnt secretion and the mRNA expression of genes interacting with the regulation of Wnt proteins, Fzd-receptors, LRP5/6 co-receptor, PKC, p38/JNK or Rho/MAPK, and cAMP–PKA cascade. Moreover, the present study confirms the implication of p38/JNK and Rho/MAPK signaling pathways in the fenoterol-induced sensitizing effect 2-adrenoceptor stimulation 2-receptor This study shows that fenoterol, a β2-adrenoceptor provokes changes in receptor conformation that results in the dissociation of heterotrimeric G proteins into Gsα and Gsβγ subunits leading to the stimulation of cAMP−PKA cascade and the G protein-coupled receptor kinases which mediate receptor desensitization. However, in the last decade, this classical model became incomplete 2-adrenoceptor stimulation can switch coupling from Gs to Gi heterotrimeric proteins or activate intracellular signaling pathways  via β-arrestin, a G protein-coupled receptor kinase α/0 subunit can also interact with the LRP5/6-Fzd receptor complex thus activating the canonical Wnt pathway α and Gβγ subunits have been shown to promote canonical Wnt signaling via Dsh, a protein involved in the Wnt/β-catenin pathway by interacting with the initiation of LRP5/6 phosphorylation by the axin-GSK-3β complex . Given that measurement of mRNA expression could not exactly reflect the changes of Wnt excretion, our results should be confirmed at the protein expression level, and extrapolating our data to clinical outcomes was beyond the scope of our study. However, it is uncertain that investigating the Wnt excretion during fenoterol incubation for 15 hours at 37°C could validate our functional results because of the action of the epithelial peptidases, limiting the half life of excreted proteins Collectively, our pharmacological investigations indicated that fenoterol may activate, at least in part, the Wnt/β-catenin signaling pathway in human isolated bronchi, suggesting a mechanism of biased agonism. To a clinical point of view, these findings highlight the interest of Wnt signaling pathways in inflammatory airway diseases. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.


In the petiole cell walls of “747” and the F1 and F2 plants that formed splitting heads, the cellulose microfibrils were loose and had separated from each other. These findings verified that anomalous cellulose microfibrils, larger cell size and thinner-walled epidermis cells are important genetic factors that make cabbage heads prone to splitting.Cabbage belonging to Brassicaceae family is one of the most important vegetables cultivated worldwide. The economically important part of cabbage crop is head, formed by leaves which may be of splitting and non-splitting types. Cabbage varieties showing head splitting causes huge loss to the farmers and therefore finding the molecular and structural basis of splitting types would be helpful to breeders. To determine which anatomical characteristics were related to head-splitting in cabbage, we analyzed two contrasting cabbage lines and their offspring using a field emission scanning electron microscope. The inbred line “747” is an early head-splitting type, while the inbred line “748” is a head-splitting-resistant type. The petiole cells of “747” seems to be larger than those of “748” at maturity; however, there was no significant difference in petiole cell size at both pre-heading and maturity stages. The lower epidermis cells of “747” were larger than those of “748” at the pre-heading and maturity stages. “747” had thinner epidermis cell wall than “748” at maturity stage, however, there was no difference of the epidermis cell wall thickness in the two lines at the pre-heading stage. The head-splitting plants in the F Brassica oleracea var. capitata L.) is one of the many varieties of Brassica oleracea, which is in the Brassicaceae family. Brassicas are important to humankind for their economic, nutritional, and potential anticancer value. The economic value of cabbages is reduced when their heads split or crack, and when they are attacked by insects.Cabbage  [Expansins are proteins that loosen the cell wall by weakening the noncovalent network formed by cellulose, hemicellulose, and pectins . Expansi (FESEM) . The resAlthough cabbages susceptible to head-splitting were found, no detail study at molecular or at anatomical structure has been done. Previous study in splitting type cabbage genotypes at maturity stage reported to observe a large leaf mesophyll cells with a loose structure . Further2 population was obtained from bud self-pollination of a single hybrid F1, which was derived by crossing “747” and “748”. The two diverse cabbage inbred lines and the F1 and F2 population were used to observe anatomical characteristics by FESEM. Thirty seeds of each parental line with F1 and F2 were germinated at the 21st February 2014 in 40-well multipots. After one month, the seedlings were transferred to 25-cm diameter pots. Plants were watered carefully onto the soil directly to avoid any possible contamination or damage to the epidermal wax [The inbred line “747” is an early head-splitting line, while the inbred line “748” is a head-splitting-resistant type . Detailsrmal wax . Leaf sa1 and F2 plants at the pre-heading and maturity stages using a single edge blade. At the pre-heading stage, the fourth leaf from the terminal bud was collected from 2-month-old seedlings of “747” and “748”. At maturity, the outer leaf of the head was collected from mature plants of the parental line, F1 and F2 plants of head-splitting and non-splitting types. At least three to five biological repeats were taken for observing anatomical characteristics from each sample. Two different methods were used to prepare samples for observations of epidermal wax and cellular structure. To observe the epidermal wax structure, fresh leaf samples were cut into small pieces, quickly frozen using liquid nitrogen, and then freeze-dried  for 24 h. For cellular structure observations, we used the method described in our previous study [2. All specimens were sputter-coated with platinum for 100–150 s and photographed under the FESEM at 10–20 kV.The anatomical characteristics of different leaf samples were observed under a FESEM . Leaf and petiole specimens were collected from individuals of “747”, “748”, and the Fus study . Fresh s2) of “747” and “748” were not significantly different at the pre-heading and maturity stages, and there was no significant difference in cell numbers between head-splitting and non-head-splitting individuals in the F1 and F2 population  Petiole cross-section; (B) enlargement of weak point from (A); (C) enlargement of normal cell structure from (A).(TIF)Click here for additional data file.S2 Fig(A) Petiole cross-section; (B) enlargement of weak point from (A); (C) enlargement of normal cell structure from (A).(TIF)Click here for additional data file.


Rats were administered 6-OHDA and 1 week later given EX-4 alone, with EX-(9-39) or nafadotride (D3 antagonist) and BrdU. Seven days later, rats were challenged with apomorphine to evaluate circling. Extracellular DA was measured using striatal microdialysis and subsequently tissue DA measured. Tyrosine hydroxylase and BrdU were verified using immunohistochemistry. Apomorphine circling was reversed by EX-4 in lesioned rats, an effect reduced by EX-4, while both EX-(9-39) and NAF attenuated this. 6-OHDA decreased extracellular and tissue DA, both reversed by EX-4 but again attenuated by EX-(9-39) or NAF. Analysis of BrdU+ cells in the SVZ revealed increases in 6-OHDA-treated rats which were reversed by EX-4 and antagonised by either EX-(9-39) or NAF, while in the SN the opposite profile was seen.Glucagon-like peptide-1 receptor (GLP-1R) activation by exendin-4 (EX-4) is effective in preclinical models of Parkinson's disease (PD) and appears to promote neurogenesis even in severely lesioned rats. In the present study, we determined the effects of EX-4 on cellular BrdU incorporation in the rat subventricular zone (SVZ) and substantia nigra (SN). We also determined the specificity of this effect with the GLP-1R antagonist EX-(9-39) as well as the potential role of dopamine (DA) D Evidence of its potential for the treatment of these neuropathologies has been accumulated rapidly [Evidence suggests that the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4), used in the treatment of type 2 diabetes mellitus, also displays neuroprotective properties in multiple cellular and  rapidly . The dru rapidly .in vitro to be GLP-1R dependent by the use of the GLP-1R selective antagonist EX-(9-39) and also more recently in GLP-1R knockout mice [EX-4 is a potent agonist at mammalian GLP-1Rs and promotes insulin secretion from beta islet cells. Additionally, EX-4 protects beta cells from cytotoxic insults  and alsoout mice . A possi3 receptors, which are thought to be involved in stimulation of neurogenesis, as demonstrated in the rat 6-OHDA model of PD [3 receptors has been shown to protect DA neurons against 6-OHDA-induced damage and promote neuronal survival as well as stimulate neurogenesis in the subventricular zone (SVZ) [3 receptors in EX-4-mediated neuroprotection by using nafadotride (NAF), a selective D3 receptor antagonist. Finally, we wished to determine the effects of EX-4 on BrdU+ cell numbers in the SVZ and SN of 6-OHDA-lesioned rats and how this might be altered by EX-(9-39) or NAF.In the context of PD, a potential influence could also be activation of DA Del of PD , 11. We ne (SVZ) , 11, we All drugs and reagents were purchased from Sigma-Aldrich Ltd., UK, unless otherwise specified.Male Wistar rats (250–300 g) were purchased from Harlan, UK, and housed under standard conditions in an animal care facility with controlled temperature, humidity, and fixed light/dark cycles. Animals were group housed and had free access to food and water. All experimental procedures were carried out in accordance with institutional and home office regulations  and the Helsinki Agreement.2 for induction (1.5% for maintenance) and then secured on a stereotaxic frame . Injection was made using a 10 μL Hamilton syringe. Prior to surgery, animals were injected with pargyline (50 mg/kg) and desipramine (25 mg/kg) intraperitoneally (i.p.). This was done to minimize the metabolism of 6-OHDA and to preserve the noradrenergic system, respectively. The animals were, then, injected with 6-OHDA hydrochloride (8 μg/2 μL) dissolved in saline containing 0.2% ascorbic acid into the left medial forebrain bundle . Infusion was performed at a rate of 1 μL per minute, and a 5-minute period was allowed for diffusion of the toxin after which the syringe was slowly retracted.Rats were anaesthetised using 5% isoflurane  v/v in Oμg/kg) or saline twice a day (10 am and 5 pm), for a period of one week, seven days after the administration of 6-OHDA. Rats cotreated with EX-9-39 received i.p. injections of the compound 1 μg/kg or saline twice a day 5 min prior to EX-4 injection. In another group of rats NAF was given i.p. (1 mg/kg), a dose that is very largely D3 selective, or saline twice daily, also 5 min prior to EX-4. The dose of NAF was based upon studies also using this dose with apparent D3 selectivity [Animals were given EX-4 i.p.  in saline, and after 15 minutes each rat was placed in a circular test arena. Two minutes later, the number of turns was counted per 120-second period. The number of turns is indicative of the presence of the neurotoxic lesion. We have used longer assessment periods in the past but found that these simply revealed quantitatively larger but qualitatively very similar numbers of turns between groups and were, therefore, deemed an unnecessary use of time. This accords with procedures we have tested and used previously .2 and 1.5% maintenance) and then secured in a stereotaxic frame. Microdialysis probes, constructed as described previously and with a membrane length of 4 mm , were bilaterally implanted into the striatum ; dental screws were placed within but not penetrating the skull; the assembly was fixed solidly with dental acrylic . After surgery, animals were placed in individual microdialysis cages and allowed to recover for 24 hours before dialysis. The following day rats were perfused with artificial cerebrospinal fluid (aCSF)  pH 7.4 at a rate of 1 μL/min using Harvard Apparatus model 22 syringe infusion pumps. After a stable baseline was established, around 1 hour, samples were collected every 30 minutes and after sample four “normal” aCSF was switched to one containing 100 mM KCl, with Na+ concentration reduction to maintain osmolarity, for 30 minutes before returning to normal aCSF for the rest of the experiment. Samples were frozen immediately at −80°C and analysed for DA within one week using high-performance liquid chromatography (HPLC) with electrochemical detection [Surgery was performed in these rats 14 days after 6-OHDA injection. Animals were anaesthetised with isoflurane  connected to a Triathlon refrigerated (4°C) autosampler  and a C18 reverse phase column maintained at 35°C  and protected by a Microsorb guard column . Detection was made with an Antec-Intro electrochemical detector  fitted with a VTO3 flow cell (Vcell +625 mV filtered to 5 abu with range set on 0.5 nA/volt). Data capture was achieved and analysed by a PC system  equipped with Chromperfect for Windows software . DA peak areas were converted to amounts using the external standard method. 2 in order to minimise stress and decapitated, and the brains were rapidly removed, flash frozen on dry ice, and stored at −80°C until use. One day before histology, frozen brains were taken from −80°C storage and stored at −20°C overnight to facilitate dissection and cryostat sectioning. Striata were removed using a stainless steel punch, placed into microcentrifuge tubes containing 1 mL ice cold 0.01 M PBS and homogenised for DA determination. Homogenates were centrifuged at 9000 ×g for 15 minutes, and duplicates of 50 μL aliquots of the supernatant were treated with 0.2 M perchloric acid containing ascorbic acid (0.2 μM) and EDTA (0.2 μM) to precipitate the cell debris. The 50 μL homogenate and 10 μL perchloric acid mixtures were centrifuged again at 9000 ×g for 15 minutes at 4°C, and 25 μL of this supernatant was injected in the HPLC system to determine tissue DA concentration in pg/gram of striatal tissue.Animals were always sacrificed on completion of microdialysis (two weeks after 6-OHDA surgery). Rats were lightly anaesthetised with 5% isoflurane v/v in Oμm thick cryostat sections from frozen brain blocks were removed from the freezer and allowed to equilibrate to room temperature for 30 minutes before fixation in 4% w/v paraformaldehyde containing 1% w/v glutaraldehyde in deionised water for 5 minutes at 0°C. Following rinsing in 0.01 M PBS for 5 minutes, sections were dehydrated through graded ethyl alcohol concentrations, and endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 in methanol for 10 minutes. The sections were then rehydrated, and nonspecific immunoreactivity was blocked using 10% swine serum in 0.01 M. PBS for 10 minutes. Sections were then incubated in primary antibody (anti-TH IgG raised in rabbit at 1 : 1000 in 0.01 M PBS) for 15 hours at 4°C. After rinsing, the sections were incubated sequentially in biotinylated swine anti-rabbit antibody 1 : 500 in 0.01 M. PBS and ABC (Vector Labs Ltd.) complex was applied for 30 minutes at room temperature following the manufacturer's instructions. Immunoreactivity was visualized through incubation in 0.5 mg/mL 3′,3-diaminobenzidine (DAB), containing 0.009% H2O2 in 20 mL of DAB solution, for 2 minutes at room temperature. The sections were counterstained in Harris haematoxylin, dehydrated, cleared, and mounted for examination. Sections were viewed under a light microscope using 5x magnification. Digital images were captured using a Leica DC500 system and the manufacturer's software.Slide mounted 12 μm thick cryostat sections from frozen brain blocks were removed from the freezer and allowed to dry for 24 hours before fixation in 75% acetone and 25% absolute alcohol for 5 minutes. Slides intended for BrdU immunodetection were also preincubated in 2 M HCl for 30 min at 37°C to facilitate antigen retrieval. The same procedure was followed as for TH immunodetection. The BrdU antibody used was a goat monoclonal raised against BrdU (1/1000) followed by a secondary rabbit anti goat-polyclonal antibody with a biotin tag (1/500). BrdU staining was quantified, by an investigator unaware of the treatment protocol, by manually counting cells over a set distance along the lateral ventricular wall viewing sagittal sections. For BrdU histology, slide mounted 12 Data obtained from apomorphine tests, tissue DA, TH, and BrdU immunohistochemistry were subjected to one-way analysis of variance (ANOVA) and post hoc Bonferroni multiple comparison tests to compare difference between selected treatments. Microdialysis data were subjected to a two-way ANOVA with a post hoc Bonferroni multiple comparison test. In both cases, data were expressed as mean ± standard error of the mean. GraphPad Prism 5 software was used for all of the statistical manipulations. Apomorphine-induced circling was tested in treated rats . The 6-OIn sham groups the mean DA content was 6.8 ± 1.1 pg/g of tissue while in 6-OHDA-only groups there was a substantial reduction in DA levels (0.77 ± 0.2 pg/g). In groups cotreated with EX-4 tissue DA was restored to 5.2 ± 0.33 pg/g, an effect reversed by both EX-9-39 (0.19 ± 0.1 pg/g) and, to a lesser extent at the doses employed here, by NAF (2.6 ± 0.53 pg/g); both results were significantly different from 6-OHDA + EX-4 . Animals+-evoked striatal extracellular DA were assessed using in vivo microdialysis, with DA release shown as fmol/10 μL of sample. Basal DA levels in control groups were 39 ± 1.2 fmol/10 μL, increasing to 1108 ± 113 fmol/10 μL when stimulated with 100 mM KCl and rapidly returning to baseline on removal of this stimulus  and K+-evoked (780 ± 87 fmol/10 μL) extracellular DA  and quantitatively Figures . SN regiBrdU was coadministered to treatment groups to investigate cell proliferation in the rat SVZ and SN while DAB staining was used to visualise BrdU antibody binding. Results were expressed as numerical density calculated as numbers of DAB positive cells per section. In the SVZ, 6-OHDA-only-treated animals showed a significant increase in BrdU+ cells, which was reversed by EX-4. In turn, the effects of EX-4 were markedly attenuated by EX-(9-39) Figures  and 6. Iin vitro and now in vivo, strongly indicate the therapeutic potential of EX-4 in neurodegenerative disorders where GLP-1Rs appear to be the key in mediating these effects. The present report indicates that GLP-1Rs as well as DA D3 receptors are required for neuroprotective effect of EX-4 in the 6-OHDA rat model of PD. Thus, when the specific antagonist of GLP-1R EX-(9-39) was coadministered with EX-4 in 6-OHDA rats, animals displayed increased contralateral circling, reduced extracellular as well as tissue DA levels, and also decreased SNc TH expression, a complete reversal of the previously observed effects of EX-4 [Many studies, both  of EX-4 .de novo neurogenesis as indicated by the findings of Bertilsson et al. [GLP-1Rs are also expressed throughout the central nervous system, with binding sites identified in both the human and the rat brain , and areIn our studies we have observed increased accumulation of BrdU+ cells in the SVZ of 6-OHDA-treated animals, an effect which was reduced significantly when animals were given EX-4, while in the SN the opposite effect was seen with reduced BrdU+ cells in 6-OHDA-treated rats and a reversal with EX-4. The effects of EX-4 were themselves completely reversed by EX-(9-39), indicating a GLP-1 receptor specific primary action. 3 receptors play a role in the overall process. D3 receptors are localized on DA neurons of the adult SN [3 receptor stimulation is neuroprotective [3 antagonist A-437203 and also in mice lacking D3 receptors [in vitro and in vivo studies suggests that protective effect of pramipexole, which is a preferential D3 agonist used in the treatment of early PD, is dependent on brain-derived neurotrophic factor (BDNF) signaling [In vivo evidence demonstrates that infusion of a D3 preferring agonist into lateral ventricles doubled cell proliferation in the SVZ and the olfactory bulb whereas no such effect was observed with a D1 preferring agonist or in the presence of a D3 antagonist [3 receptor and was reduced in the presence of an antagonist of this receptor subtype [3 receptor stimulation not only results in SVZ cell proliferation but stimulated cells also display neuron specific markers in both SVZ and the rostral migratory stream [3 preferring agonist into the third ventricle resulted in a sixfold increase in BrdU-labeled cells in the SN which were also TH positive and displayed a neuronal phenotype [3 receptors has not been studied directly as far as we are aware but is of obvious interest and has been tentatively suggested [3 receptor blockade [Additionally, the effects of EX-4 in the SVZ and SN were attenuated by NAF, indicating that DA Dadult SN , 11 as wadult SN , 11, 18.otective . Selectiotective , 20 and otective , 11, 21.eceptors . Evidencignaling , 23, 24.tagonist , 11. In  subtype . D3 recey stream , 11. Moruggested . Our chouggested , 13 wherThe effects of 6-OHDA on BrdU incorporation into cells in the SVZ are themselves somewhat controversial , 27. Pro3 receptor stimulation one possibility may be that EX-4 alters the migration and/or maturation of progenitor cells. Moreover, our findings with EX-9-39 unambiguously show that these effects are primarily mediated by the GLP-1R.EX-4 has proven efficacy in preclinical models of PD. More recently evidence suggests that EX-4 has actual therapeutic efficacy, as indicated in a small scale clinical trial . These o