The chemokine receptors CX3CR1 and CCR2 have been implicated in the development of age-related macular degeneration (AMD). The evidence is mainly derived from experimental cell studies and murine models of AMD. The purpose of this study was to investigate the association between expression of CX3CR1 and CCR2 on different leukocyte subsets and AMD. Furthermore we measured the plasma levels of ligands CX3CL1 and CCL2.Patients attending our department were asked to participate in the study. The diagnosis of AMD was based on clinical examination and multimodal imaging techniques. Chemokine plasma level and chemokine receptor expression were measured by flow-cytometry.+ T cells in the neovascular AMD group compared to the control group (p = 0.04). We found a significant positive correlation between CCR2 and CX3CR1 expression on CD8+ cells . We found no difference in plasma levels of CX3CL1 and CCL2 among the groups.A total of 150 participants were included. We found a significantly lower expression of CX3CR1 on CD8+ cells; this correlated to a low expression of CCR2 on CD8+ cells. Further studies are needed to elucidate the possible role of this cell type in AMD development.Our results show a down regulation of CX3CR1 on CD8 Age-related macular degeneration (AMD) is a disease with a complex etiology. Inflammation is thought to be a major factor in development and progression of AMD.  al. showed that the lesions found in CX3CR1 knock-out mice consisted of lipid bloated macrophages that accumulate subretinally probably due to deficiencies in the migratory properties. CCL2 and CX3CL1 signaling through CCR2 and CX3CR1 have shown to be key factors in recruitment of macrophages to tissue lesions or sites of inflammation. There is increasing evidence of CCR2 and CX3CR1 and their ligands, CCL2 and CX3CL1, being involved in the development and progression of AMD. Since most of the studies made in this area are carried out on mouse models of AMD and in experimental cell studies, we examined the peripheral expression of CCR2 and CX3CR1 on different lymphocyte subsets and measured the plasma levels of CCL2 and CX3CL1 in patients with AMD.During a period of 20 months, patients with AMD attending our department were asked to participate in this case-control study. Individuals attending our department for other reasons were asked to participate as control subjects. AMD was diagnosed according to the Age-Related Eye Disease Study (AREDs) criteria. Participants were excluded from the study if they were diagnosed with malignant or autoimmune disease including type 1 diabetes. All participants in active treatment with immunosuppressive agents were excluded. To avoid interference from other acute-phase responses due to undiagnosed cancers and acute infections serum C-reactive protein (CRP) was measured in all participants. All participants having a serum level of CRP>10 mg/L were excluded.The participants included were divided according to their AMD stage in the following groups: 1) Healthy controls; 2) Early AMD defined by the presence of drusen and 3) Neovascular AMD. None of the participants had signs of polypoidal vasculopathy, retinal angiomatous proliferation, or chorioretinal anastomosis. None of the participants had reticular drusen. All patients diagnosed with neovascular AMD were treatment naïve to Bevacizumab , Aflibercept  and had not received injection with Ranibizumab  for the last five weeks prior to inclusion.All participants underwent a structured interview with focus on current and previously medical conditions and current medication, smoking habits and alcohol consumption. Patients were defined as current smokers, former smokers (more than 100 cigarettes during their lifetime) or never smokers. Alcohol consumption was graded according to the Danish National Board of Health’s recommendations . Height and weight were measured at the first visit to calculate the body mass index (BMI).Part of the study population described in this study has been described in previous studies.Verbal and written informed consent was obtained from all participants prior to inclusion. The study has been approved by the Regional Committee of Ethics in Research of the Region of Zealand (SJ-142) and was performed in adherence to the Declaration of Helsinki.All participants underwent a thorough ophthalmologic examination including slitlamp biomicroscopy and fundoscopy, colour fundus photography , Spectral-Domain-Optical Coherence Tomography (SD-OCT) and fundus autofluorescence imaging . Fluorescein and indocyanine green (FA/ICG) angiography was performed on patients suspicious of neovascular AMD in order to obtain correct diagnosis. Visual acuity (VA) was measured using the Early Treatment Diabetic Retinopathy Study (ETDRS) chart. Venous blood samples were obtained from all participants on the day of inclusion and the blood samples were prepared for flow cytometry within 4 hours of phlebotomy. In patients undergoing retinal angiography, phlebotomy was performed prior to angiography in order to avoid possible interference. Heparinized blood samples were centrifuged for 15 minutes at 1000 G, and plasma was isolated and stored at −80°C until analysis were performed. CX3CL1 and CCL2 were measured on an LSR II flow cytometer using a Cytometric Bead Array  following the manufacture’s recommendations.To assess the clinical relevance of peripheral expression of CCR2 and CX3CR1 and CX3CL1 and CCL2 we looked at treatment response after the initial three anti-VEGF injections with Ranibizumab. It has been suggested, that VA at this point of treatment is a useful predictor (among others) of treatment response. + T cells. The study population was divided in tertiles based on the expression of CX3CR1 and CCR2 on CD8+ cells. Odds Ratios (OR) for the prevalence of AMD were calculated by comparing the highest and lowest tertile for each receptor. A p-level of less than 0.05 was considered significant. A power calculation showed that a sample size of 24 in each group was needed to reach a power level of 80% and a statistical significance level of 0.05.The statistical software SPSS version 20 for Windows  was used for statistical analysis. Kruskal-Wallis test was used for one-way analysis of variance and Mann-Whitney U test was used to compare 2 groups following analysis of variance for not normally distributed continuous variables  and data are given as medians and interquartile ranges (IQR). Categorical variables were analyzed using Pearson Chi-square test . Spearman correlation coefficient was used to examine the relationship between expression level of CCR2 and CX3CR1 on CD8A total of 150 participants were included in the study. Of these, 90 patients had neovascular AMD and 30 patients had early AMD. Thirty age-matched control persons were included. The clinical and demographic data can be seen in + and CD8+ T cells. The data is shown in + T cells expressing CX3CR1 in the neovascular AMD group compared to the control group . Similarly, we found a tendency to lower frequency of CD8+ T cells expressing CCR2 in the group with neovascular AMD compared to the control . We found a significant positive correlation between percentage of CD8+ CCR2+ cells and CD8+ CX3CR1+ cells . To see whether the AMD prevalence was influenced by expression of CX3CR1 and CCR2 on CD8+ cells, we divided the study population in tertiles according to frequency of CCR2 and CX3CR1 expression on CD8+ cells as previously described. We found that patients with CX3CR1 expression in the lowest tertile had a crude OR for AMD (early and late stage) prevalence of 5.3  1.1–25.2). +CCR2+ frequency against CD8+CX3CR1+ frequency. Combined CCR2 and CX3CR1 receptor expression data was available in 104 patients. The plot shows that almost all of the study persons with combined low frequency of CCR2 and CX3CR1 on CD8 cells were diagnosed with AMD.CX3CR1 is expressed on a subset of T cells and is important for the cells ability to migrate to sites of inflammation. high CX3CR1low 2. Intermediate (pro-inflammatory) monocytes being CCR2mid CX3CR1high and 3. Non-classical (patrolling) monocytes being CCR2low CX3CR1high. The data is shown in Monocytes can be differentiated in different functional phenotypes according to their expression of CX3CR1 and CCR2. The plasma levels of CX3CL1 and CCL2 were measured in all participants in the study. CX3CL1 is a chemokine found in the blood as membrane bound to different cell types but also as a soluble non-bound protein. + cells in the group with a VA loss of more than 10 ETDRS letters even though no significant change was found between the groups   .Age-related macular degeneration is a disease with a complex etiology. Inflammation and dysregulation of inflammatory responses play an important role in development and progression of AMD. It is believed that the main function of CCR2/CCL2 and CX3CR1/CX3CL1 is to recruit monocytes to sites of injury and inflammation.  al. found that CCL2−/− and CCR2−/− knock-out mice develop many of the features found in early AMD. Thickening of Bruch’s membrane, swelling of the RPE cells and drusen-like lesions were found in the knock-out mice before the wild type mice developed these features.  al. found that intraocular levels of CCL2 was increased in patients with neovascular AMD. + inflammatory infiltrating monocytes are increased in patients with geographic atrophy. −/− knock-out mice, Luhmann et al. found that the there was an age-related accumulation of bloated macrophages subretinally and that this process was accelerated in the CCL2−/− knock-out mice model supporting previous findings by Xu et al. who found an age dependent subretinal accumulation of microglia, suggesting that there is a normal age-related increase in subretinal accumulating cells.  al. did not find any spontaneous CNV formation in the ageing mice suggesting that the lack of CCL2 does not lead to CNV development. This is further supported by Tsutsumi et al. who found that laser-induced CNVs were smaller in CCR2−/− knock-out mice compared with wild type mice.  al. showed that intravitreal injections with a CCR2 antagonist significantly could reduce the size of laser-induced CNV in mice. Moreover, the number of macrophages infiltrating the choroid decreased significantly in mice treated with the CCR2 antagonist, as well as a decrease in VEGF expression was seen in the treated mice. Substantial evidence, points towards the CCR2/CCL2 receptor/ligand complex as an important factor in the development of AMD, even though contradictory results have come forward. Ambati et+ cells in the neovascular AMD group. CCL2 is not expressed at very high levels in the retina under normal conditions.  al. reported a few years back that CCR2 and CCR1 was increased on a sub-type of monocytes in peripheral blood in patients with neovascular AMD. We measured the expression of CCR2 on different subsets of leukocytes but did not find any significant differences between the groups of participants. Nevertheless, there was a tendency towards a lower expression of CCR2 on CD8 al. proved the migratory properties of microglia in the retina of albino mice strains. −/− knock-out mice microglia cells accumulate in the subretinal space with age after laser injury. The extended presence of microglia cells in the subretinal space results in excessive phagocytosis of outer segments, and the drusen-like lesions seen in CX3CR1 deficient mice have been found to be lipid bloated macrophages. + cells expressing CX3CR1 compared to healthy control persons. A lower expression of CX3CR1 is, as described previously, influencing the lymphocytic migratory properties. Down regulation of CX3CR1 expression could imply decreased ability of cells to migrate to sites of inflammation. This could be potentially beneficial since the recruitment of mononuclear cells to the subretinal space could lead to neurotoxic impact on the retina. At the same time lack of migratory properties could also trigger the inflammatory response in the retina because of the impaired trafficking of microglia cells within the retina. We measured the plasma level of CX3CL1 but did not find any difference between patients with AMD and control persons.Like CCR2, the chemokine receptor CX3CR1 is important for the migration of leukocytes to sites of inflammation. Ng et+ cells expressing CX3CR1 had a higher OR for developing late stage AMD compared to healthy controls. We found that the majority of patients having AMD and a low fraction of CX3CR1 expressing CD8+ cells, also had a low fraction of CD8+ cells expressing CCR2. A CCL2−/− CX3CR1−/− mouse model of AMD has been suggested, however contradictory findings have been published. We found that patients with low percentages of CD8+ cells in the group of patients with neovascular AMD who lost more than 10 ETDRS letters after the initial three anti-VEGF injections. It is possible that higher expression of CCR2 leads to a more pro-angiogenic environment, which in turn could result in a worse response to anti-VEGF treatment. It has been described that tissue expression of VEGF increases with increased CCR2 expression, and a synergistic effect on angiogenesis has been suggested.  al. also looked at the clinical relevance of CCR2 and CCR1 expressed on a subset of monocytes and did not find any relationship between chemokine receptor expression and treatment response. We found a tendency towards higher expression of CCR2 on CD4The divergence in the reports about CCR2/CCL2 and CX3CR1/CX3CL1 involvement in development and progression of AMD is most likely a reflection of the complexity of the pathogenesis of AMD. The knock-out mice strains and murine models of AMD represents very regulated environments in contrast to the aging human body. In addition, chemokine receptors can be altered locally without this being reflected systemically. Therefore, our findings do not rule out CCR2 as a local factor in development of AMD, but indicates that this alteration in expression in the eye is not reflected systemically. Furthermore, it is unlikely that dysregulation of a single receptor or ligand can explain the development of AMD.+ cells in patients with neovascular AMD compared to control persons, and found that most of the patients with low expression of CX3CR1 and CCR2 on CD8+ T cells were diagnosed with AMD.In conclusion, different murine models and experimental studies have revealed the involvement of CCR2 and CX3CR1 in the development and progression of AMD. Our findings also suggest a possible role for those chemokines in AMD pathogenesis, since we found a lower fraction of CX3CR1 expression on peripheral CD8


The liver has a large regenerative capacity. Hepatocytes can replicate and regenerate a diseased liver. However, as is the case in severe liver diseases, this replication may become insufficient or exhausted and hepatic progenitor cells (HPCs) can be activated in an attempt to restore liver function. Due to their bi-potent differentiation capacity, these HPCs have great potential for regenerative approaches yet over-activation does pose potential health risks. Therefore the mechanisms leading to activation must be elucidated prior to safe implementation in the veterinary clinic. Wnt/β-catenin and Notch signalling have been implicated in the activation of HPCs in mouse models and in humans. Here we assessed the involvement in canine HPC activation. Gene-expression profiles were derived from laser microdissected HPC niches from lobular dissecting hepatitis (LDH) and normal liver tissue, with a focus on Wnt/β-catenin and Notch signalling. Immunohistochemical and immunofluorescent studies were combined to assess the role of the pathways in HPCs during LDH.Gene-expression confirmed higher expression of Wnt/β-catenin and Notch pathway components and target genes in activated HPC niches in diseased liver compared to quiescent HPC niches from normal liver. Immunofluorescence confirmed the activation of these pathways in the HPCs during disease. Immunohistochemistry showed proliferating HPCs during LDH, and double immunofluorescence showed downregulation of Wnt/β-catenin and Notch in differentiating HPCs. Vimentin, a mesenchymal marker, was expressed on a subset of undifferentiated HPCs.Together these studies clearly revealed that both Wnt/β-catenin and Notch signalling pathways are enhanced in undifferentiated, proliferating and potentially migrating HPCs during severe progressive canine liver disease (LDH). Liver diseases occur frequently in the canine pet population. Around 12% of the dogs in first opinion practices have liver disease . It is cIrrespective of the cause of hepatocyte damage, the liver can recover from such insults due to replication of fully differentiated hepatocytes . In caseIn order to safely use HPCs for liver regeneration in a clinical setting where hepatocyte replication is insufficient, detailed knowledge of crucial signalling cascades for HPC activation is essential. Two signalling pathways, Wnt/β-catenin and Notch, are involved in proliferation and differentiation of progenitor cells including HPCs in other mammals -20. ThisThese data show that both Wnt/β-catenin and Notch signalling are enhanced in activated HPC niches in dogs with LDH. The previous descriptions of the cellular constituents of the canine HPC niche and the current investigation on specific signalling cascades clearly show the similarities with other mammals, including human ,13. It iFZD1 and the Wnt-induced transcription factor TCF3 were significantly higher in LDH cases compared to normal controls, as measured in LMD samples  stained with Keratin(K)7 (marker of HPCs) are depicted in Figure To specify the cellular origin of the upregulated gene expression levels, double immunofluorescence for β-catenin/K7 Figure A and NotTo investigate the functional involvement of Wnt and Notch in HPC activation, immunohistochemical and immunofluorescent stainings were performed Figure . With KiIn the present study the involvement of the Wnt/β-catenin and Notch pathways was investigated in canine hepatic progenitor cell activation in LDH, a highly fibrotic and progressive liver disease. The combination of laser-microdissection, gene expression studies (Q-PCR) and immunofluorescence showed the enhanced signalling of the Wnt/β-catenin and Notch pathways in activated HPC niches compared to quiescent HPCs in normal liver. This extends the findings in mouse and human liver cancer and normal liver regeneration ,21-27. TThe effect of Wnt/β-catenin was previously specified as (induction of) proliferation in rodent models of HPC activation ,24,29,30in vitro Wnt activation leads to hepatic specification [in vitro in more committed multipotent cells, active Wnt inhibits (further) hepatocyte differentiation but rather guides cells to the biliary phenotype [NOTCH1 and NOTCH3 expression is increased, HPC differentiation towards hepatocytes is inhibited, while bile duct differentiation may be enhanced. This is corroborated by the immunofluorescence stainings, where Notch1/NICD is lost with differentiation.Besides the described role in proliferation and migration, both pathways can be involved in HPC differentiation. The influence of Wnt on cell fate determination is time and place dependent. During early embryonic development and in pluripotent embryonic stem cells fication ,41. Latehenotype ,43. Regahenotype , displayhenotype . Unfortuhenotype ,45. The henotype ,46-51. Mhenotype ,53. Convin vitro studies.The activated states of the Wnt and Notch pathway in the diseased tissue were found at the same histological location, suggesting that Wnt and Notch act simultaneously. In view of this, the Wnt and Notch pathways may be intertwined in the activation of HPCs during liver disease, as occurs for example when cell-fate decisions are made during development . Whetherin vitro experiments with canine hepatic progenitor cells, at present not available, could shed light on these questions separately. These data from a non-experimental liver disease in client-owned pets confirm the previous separate reports on Wnt and Notch signalling in rat and mouse injury models of liver disease [The combined Q-PCR and immunofluorescence results extend existing literature on other species and indicate a critical role for Wnt and Notch in proliferation, differentiation and/or migration of canine HPCs during rapidly progressing fibrotic liver disease with hampered hepatocytic proliferation. The descriptive data presented here suggest a role in HPC activation;  disease ,29,30,50 disease . In the Liver samples were obtained from privately owned dogs with LDH , a rapidly progressing disease characterized by diffuse inflammation, peri-cellular fibrosis and massive HPC activation ,13. Info6 μm2 tissue was laser-dissected using four tissue sections per normal sample and two tissue sections per diseased sample.Cryosections (10 μm) were cut using RNAse free blades on a cryostat at -20°C , mounted on a pre-cooled (4°C) RNAse-free poly-ethylene naphthalene (PEN) membrane slide , immediately placed on dry ice and stored at -70°C for a maximum of one week until use. Rapid immunohistochemistry before laser microdissection (LMD) was performed with buffers and solutions prepared with DEPC-treated water . All glassware was treated with RNase Zap (Ambion) and washed with DEPC-treated water prior to use. To protect RNA from degradation during incubations at room temperature (RT), antibody and DAB solutions were prepared with 0.4 U/μl SUPERase RNase Inhibitor (Ambion). Frozen sections were taken from -70°C storage, immediately fixed in ice-cold acetone (-20°C) for 5 minutes and washed briefly (3-5 seconds) in phosphate-buffered saline (PBS). Sections were incubated with K7 antibody  in PBS for 7 minutes at RT, briefly washed in PBS and subsequently incubated in EnVision goat anti-mouse peroxidase-conjugated antibody (Dako) for 7 minutes at RT. Staining was visualised using the chromogen diaminobenzidine  for 3 minutes at RT. Finally, sections were dehydrated in an EtOH series . The LMD procedure was performed within a maximum of 20 minutes upon staining with a Nikon eclipse TE300 inverted microscope  connected to a Sony 3-CCD Microscope ColorColour Video Camera  using MMI CellTools software . Tubes with an adhesive lid (MMI) were used to remove laser dissected cells from the whole liver tissue slide. After the LMD procedure, collected cells were retrieved with 50 μl Extraction Buffer  and a short centrifugal step for further molecular processing. The cell-suspension was then incubated at 42°C for 30 min, centrifuged for 2 minutes at 800 × g, snap frozen in liquid nitrogen and stored at -70°C until further use. From each sample, a total of 2-3.5 × 10Total RNA was extracted from the LMD samples using the PicoPure RNA isolation kit  according to the manufacturer’s instructions and an on column DNAse treatment (0.1 U/μl) to remove all DNA contaminations . RNA quality after LMD was determined using a RNA 6000 Pico-LabChip with an Agilent BioAnalyzer 2100 . Amplification of the RNA was performed with the WT-Ovation™ Pico RNA Amplification System according to the manufacturer’s instructions . Making use of a DNA/RNA chimeric primer, cDNA was prepared from total RNA and amplified by linear isothermal DNA amplification. The product consists of single-strand DNA (ssDNA). The amplified product was purified with DNA Clean & Concentrator-25 from Zymo research according to the manufacturers instruction , replacing Wash Buffer by fresh 80% ethanol.B2MG, HPRT, RPS5, and RPS19.For gene expression analysis, a SYBR Green based quantitative RT-PCR (Q-PCR) was performed on a Bio-Rad My-IQ detection system as described previously  for up tRelative gene expression of each gene-product (delta-Cq method) was used as the basis for all comparisons. The non-parametric Mann-Whitney U test was performed to assess statistical differences between normal and diseased tissue using SPSS software . Gene expressions that were not detectable were arbitrarily set to Cq 45 for statistical analysis. p-Values < 0.05 were considered statistically significant.Antibody details for immunohistochemistry/-fluorescence can be found in Table 2O2 in PBS/T and background staining was blocked with 10% normal goat serum in PBS/T for 30 minutes at RT. Primary antibody was diluted in blocking serum and incubated for 60 minutes at RT. The HRP Envision system (Dako) was used. Staining was visualised using diaminobenzidine  and counter stained with haematoxylin quickstain (Vector Laboratories) for 5 minutes. Finally, slides were covered with Aquamount (Vector Laboratories). Canine duodenum served as the positive control (data not shown). Immunohistochemistry for vimentin was performed on 4 μm thick, paraffin embedded sections essentially as described previously [Whole liver cryosections (6 μm) were immunohistochemically stained for Ki67, a marker of active cell proliferation. Slides were air dried for 30 minutes at RT, fixed in ice-cold aceton:methanol 1:1 and washed in phosphate buffered saline with 0.1% Tween 20 . Endogenous peroxidase activity was blocked for 30 minutes at RT in 0.3% Heviously . ImmunohImmunofluorescent stainings were performed with parallel antibody incubations. The slides were incubated with mixed primary antibodies over night at 4°C and with mixed secondary antibodies at RT for 60 minutes. Rinsing steps were performed using TBS with 0.1% Tween 20 and slides were covered with Aquamount (Vector Laboratories). Immunofluorescent double stainings for K7/β-catenin and K7/Notch1-NICD were performed on 15 μm ice-cold acetone fixed  liver-cryosections. Immunofluorescent double stainings against PanCK/Vimentin, HepPar/β-catenin and HepPar/Notch1-NICD were performed on 4 μm paraffin embedded canine LDH liver sections essentially as described previously . For Not


Despite evidence that activated macrophages act in an inflammatory microenvironment to promote gastric tumorigenesis via β-catenin signaling, the effects of β-catenin signaling on gastric cancer cell metastasis and the relationship of these cells with surrounding tumor associated macrophages have not been directly studied.Immunohistochemical staining was employed to analyze 103 patients. An invasion assay was used to evaluate the relationship between macrophages and gastric cancer cells. β-catenin gain-of-function and loss-of-function approaches were performed. To assess the β-catenin regulation mechanism in gastric cancer cells, Western blotting and reverse-transcription polymerase chain reaction were used.Increased density of macrophages was associated with advanced stage and poor survival. Gastric cancer cell lines co-cultured with macrophages conditioned medium showed increased nuclear accumulation of β-catenin and increased invading ability. AKT but not ERK regulated β-catenin translocation. MMP7 and CD44, both β-catenin downstream genes, were involved in macrophage-activated gastric cancer cell invasion.Collectively, the clinical data suggest that macrophage infiltration is correlated with increased grade and poor prognosis for gastric cancer patients who underwent radical resection. Macrophages may induce invasiveness by activating the β-catenin pathway. Helicobacter pylori infection, which leads to chronic inflammation.[Gastric cancer (GC) is among the most common cancers worldwide and almost two-thirds of such patients will die of their disease. GC is clammation. This infammation. Researchammation., 5 HowevActivation of the Wnt pathway is an important step in carcinogenesis. Mutations along the Wnt–β-catenin pathway occur in approximately 90% of colorectal and hepatocellular carcinomas and in about 30% of GCs., 7 In adBased on above findings, we hypothesized that the β-catenin pathway may also be involved in regulating macrophage-induced GC metastasis. In this study, we first examined the density of infiltrated macrophages and the expression of β-catenin in gastric carcinoma tissues using immunohistochemistry (IHC). The clinicopathological characteristics of gastric carcinoma and prognosis were demonstrated. Furthermore, we found that macrophages induce nuclear translocation of β-catenin and enhance the invasion ability of GC cells. Our findings demonstrate and further support an important link between TAM and its downstream signaling mediator, β-catenin, in regulating GC metastasis.The study was approved by the Institutional Review Board and Human Ethics Committee of National Taiwan University Hospital. Written consent for using the samples for research purposes was obtained from all patients prior to surgery. Patient information was anonymized and de-identified prior to analysis.The study retrospectively enrolled 205 patients diagnosed with GC who received radical surgical resection at the Department of General Surgery, National Taiwan University from January 1998 to January 2002. Of these, 102 patients who lacked follow-up data or who died from perioperative complications were excluded and the remaining 103 patients were included in analyses. Clinicopathological variables were classified according to TNM classification  and the characteristics are summarized in The resected specimens of GC were fixed in formalin and embedded in paraffin. Sections were examined for TAM infiltration and β-catenin using monoclonal anti-CD68 antibody  and monoclonal β-catenin antibody , respectively.To evaluate the density of tissue-infiltrating CD68+ macrophages, tissue sections were screened by a board certified pathologist (CI Jan) at low power (100×) and the five most representative fields were selected at 400× magnification. The results were counted manually and were expressed as the sum of the number of cells in five 400× microscopic fields for each area of every specimen.GC cells AGS, N87 ), MKN45 ) and TSGH ) and human monocytic cell line THP-1 ) cells were grown in RPMI-1640 medium  with 10% fetal bovine serum  and 2 mM L-glutamine .THP-1 cells were seeded into culture dishes and induced to differentiate into macrophages by incubation with 100 ng/ml 12-O-tetradecanoylphorbol-13-acetate  for 24 h. The macrophages were washed three times with RPMI medium containing 10% FBS, incubated in this medium for another 24 h to eliminate the effect of TPA and incubated in serum-free media for another 24 h. The harvested and pooled culture supernatants were used as macrophage conditioned medium (CM) as previously described., 10GC cells were seeded in 96-well plates and co-cultured with or without macrophage CM. The proliferation/viability was determined by a colorimetric assay using 3--2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich).The invasion assay was conducted using transwell cell culture chambers . The invading cells were counted at 400× magnification in 10 different fields for each insert. The experiment was repeated three times.After stimulation with or without macrophage CM for 6 h, GC cells were collected and total RNA extracted using the Trizol reagent kit  following the manufacturer’s instructions. cDNA was amplified by polymerase chain reaction (PCR) using primers for MMP7 , CD44 , cyclin D1 , c-myc  and GAPDH .After stimulation with or without macrophage CM for 6 hours, GC cells were lysed in 300 μl lysis buffer on ice and centrifuge at 5,400 rpm. Supernatant was collected as a cytosolic fraction. Nuclear fractions were collected, then run in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for Western blot analysis.After stimulation with macrophage CM, cells were washed with PBS, scraped into RIPA buffer and centrifuged. The cell lysates were subjected to 10% SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corp.) The membrane was probed using primary antibodies for β-catenin, AKT, ERK, JNK , MMP7 , CD44 , c-myc , cyclin D1 , β-actin , HDAC1, p-AKT, p-ERK, p-JNK  and secondary antibody (Santa Cruz Biotechnology).Anti-total β-catenin antibody was used as the primary antibody, and anti-mouse Immunoglobulin G (IgG) Alexa 594 was used as the secondary antibody. Cover slips were then mounted using mounting medium containing DAPI for nuclear staining.6) into 10 cm2 dishes 1 day before transfection. Then, cells were transfected with 10 μg shRNA vectors and 10 μg of pCMVΔR8.91 (the packaging vector) and 1 μg of pMD.G (the envelope vector). After 5 h of incubation, transfection medium was replaced with fresh culture medium. Forty-eight hours later, lentivirus-containing medium was collected from transfection and spun down at 1500 rpm for 5 min to pellet the cell debris, the supernatant was filtered through a 0.45-μm filter, and target cells were infected with fresh lentivirus-containing medium (supplemented with 8 μg/ml polybrene) for 24 h.Short hairpin RNAs (shRNAs) were purchased from the National RNAi core Facility at Academic Sinica . The target sequence of β-catenin shRNA 1 was 5’- CCGGTCTAACCTCACTTGCAATAATCTCGAGATTATTGCAAGTGAGGTTAGATTTTTG-3’; that of β-catenin shRNA 2 was 5’-CCGGTTGTTATCAGAGGACT-AAATACTCGAGTATTTAGTCCTCTGATAACAATTTTTG-3’. The lentiviral vector and its packaging vectors were transfected into 293T packaging cells by calcium phosphate transfection. Briefly, 293T cells were split . All GC cell lines tested would recruit macrophages to migrate and the macrophage-recruiting ability was dependent on the degree of malignancy of the cancer cells as determined by their invasiveness Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S3 FigAKT, ERK and β-catenin protein expression were determined by Western blot.(TIFF)Click here for additional data file.S4 FigN87 cells in the presence of macrophage CM for 24 hours were pre-treated with or without TNF-α inhibitor for 1 hour.(TIFF)Click here for additional data file.


The β-catenin signaling is important in cell growth and differentiation and is frequently dysregulated in various cancers. The most well-known mechanism of endocrine resistance is cross-talk between the estrogen receptor (ER) and other growth factor signaling, such as phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) signaling pathway. In the present study, we investigated whether β-catenin could be a potential target to overcome endocrine resistance in breast cancer.We established tamoxifen-resistant (TamR) cell line via long-term exposure of MCF-7 breast cancer cells to gradually increasing concentrations of tamoxifen. The levels of protein expression and mRNA transcripts were determined using western blot analysis and real-time quantitative PCR. The transcriptional activity of β-catenin was measured using luciferase activity assay.TamR cells showed a mesenchymal phenotype, and exhibited a relatively decreased expression of ER and increased expression of human epidermal growth factor receptor 2 and the epidermal growth factor receptor. We confirmed that the expression and transcriptional activity of β-catenin were increased in TamR cells compared with control cells. The expression and transcriptional activity of β-catenin were inhibited by β-catenin small-molecule inhibitor, ICG-001 or β-catenin siRNA. The viability of TamR cells, which showed no change after treatment with tamoxifen, was reduced by ICG-001 or β-catenin siRNA. The combination of ICG-001 and mTOR inhibitor, rapamycin, yielded an additive effect on the inhibition of viability in TamR cells.These results suggest that β-catenin plays a role in tamoxifen-resistant breast cancer, and the inhibition of β-catenin may be a potential target in tamoxifen-resistant breast cancer. Breast cancer is the second most common malignancy among women in South Korea. It is a heterogeneous disease that can be classified into multiple subtypes with distinctive histological and biological features . The mosOver the past few decades, there have been many studies about the mechanisms of resistance to endocrine therapy. Although the exact molecular mechanisms underlying this phenomenon are still not completely understood, several theories have been proposed, such as the loss of ER expression, mutations within the gene that encodes the ER, adaptation of estrogen withdrawal, cross-talk with other growth factor receptor pathways, and alteration of the cell-cycle signaling pathway , 4, 5. AAberrant activation of Wnt/β-catenin signaling is observed in many human cancers, such as colon cancer . Recent In this study, we aimed to assess the expression and transcriptional activity of β-catenin in tamoxifen-resistant breast cancer cell line and evaluate the effect of inhibition of β-catenin on the viability of tamoxifen-resistant breast cancer cells.5/cm2 and cultured in phenol-red-free RPMI 1640 medium containing 10% fetal bovine serum (FBS) and antibiotics. According to a methodology reported elsewhere  (Promega) and pRL-TK constructs (Promega) using Lipofectamine 2000 . The pGL4.49 [luc2p/TCF-LEF RE/Hygro] vector contains eight copies of a TCF-LEF response element (TEF-LEF RE) that drives transcription of the firefly luciferase reporter gene luc 2p. The pRL-TK vector provides constitutive expression of Renilla luciferase as a control reporter vector. The cells were incubated for 24 hours after transfection, followed by treatment with ICG-001 or rapamycin for 24 hours. The cells were then processed using the DUAL-Luciferase Reporter Assay System (Promega), and luciferase activities were measured on a luminometer. The ratio of firefly to Renilla luciferase activity was representative of the transcriptional activity of β-catenin.Luciferase assay kit from Promega  was used. MCF-7 and TamR cells were seeded at a density of 4 × 10® β-catenin siRNA I). The mixture of β-catenin siRNA (100 nM) and Lipofectamine 2000 in Opti-MEM was incubated in room air for 20 minutes. MCF-7 and TamR cells were seeded at a density of 5 × 105 cells in 100 mm dishes and then were incubated with mixture of β-catenin siRNA and Lipofectamine 2000 in a CO2 incubator  for 48 hours. The Control siRNA  siRNA) was used as negative control (Mock).The siRNA against β-catenin was obtained from Cell Signaling Technology  and rapamycin (20nM) for 24 h for cell cycle analysis. The cells were detached with trypsin and pelleted by centrifugation at 1,200 rpm for 5 min, followed by fixation with 70% ethanol for overnight at -20°C. The cells were washed with PBS and cellular DNA was stained with 0.5 ml of protium iodide (PI) solution  for 30 min at room temperature. For annexin V staining, cells were stained with FITC annexin V in binding buffer (10 mM HEPES (pH 7.4), 140 mM NaCl, 0.25 mM CaCl2) and 5 ug/ml of PI. Analytical cytometry was performed on FACSCalibur (BD). Cell cycle analysis was performed with CellQuest (BD) and ModFit  software.Results were presented as means with error bars representing the standard deviation. Statistical analyses were performed using SPSS . An unpaired t-test was used to evaluate significant differences among the continuous variables. A P-value ≤ 0.05 was considered statistically significant.TamR cells had a markedly different morphology compared with the parental control cells . The majWe confirmed the protein expression levels of unphosphorylated transcriptionally active β-catenin using two methods: Western blot analysis using a commercial primary antibody and pull-down assay. Both methods showed that the expression of the active β-catenin protein was increased in TamR cells compared with control cells . The traICG-001 is a small-molecule inhibitor that antagonizes β-catenin/TCF-mediated transcription . TranscrTo the next, control and TamR cells were transfected with siRNA specific for β-catenin and then the protein levels of β-catenin were assessed by western blot analysis. The β-catenin siRNA suppressed the expression of total and active β-catenin . Taken tCell viability assays were conducted to determine the growth inhibitory effect of inhibition of β-catenin. TamR cells did not show a change in viability by tamoxifen (3 μM). However, ICG-001 (40 μM) reduced the viability of TamR cells by 45% and β-catenin siRNA also reduced the viability of TamR cells by 41% .PI3K/Akt/mTOR signaling pathway is a well-known mechanism of endocrine resistance. Rapamycin is the mTOR inhibitor, which previously showed overcoming endocrine resistance in breast cancer cells . To confWe evaluated the protein levels and transcriptional activity of active β-catenin after treatment with ICG-001 (40 μM), rapamycin (20 nM), and a combination of ICG-001 and rapamycin, respectively. The expression β-catenin was decreased by rapamycin as well as by ICG-001. Pull-down assays tend to measure the expression of β-catenin more accurately. The combination of ICG-001 and rapamycin led to a greater decrease in the expression and transcriptional activity of β-catenin compared with each single inhibitor .Cell cycle of control and TamR cells was analyzed by flow cytometry. The results showed that TamR cells had an acceleration of the G1 to S phase transition compared to control cells, indicating that cell cycle progression was promoted in TamR cells. ICG-001 increased apoptosis and rapamycin induced significant G0/G1 phase arrest in TamR cells . The comWe confirmed the protein levels of pAkt, pGSK-3β, and pp70S6K in TamR cells, as well as the change in their expression levels after treatment with ICG-001 (40 μM), rapamycin (20 nM), and a combination of ICG-001 and rapamycin . The expWe established tamoxifen-resistant breast cancer cell line by continuously exposing ER-positive breast cancer cells to tamoxifen by a gradual process. In the present study, TamR cells showed relatively decreased expression of ERα and increased expression of HER2 and EGFR. This result was consistent with previous research  and indiThe previous studies have reported that activation of the PI3K/Akt/mTOR pathway is associated with endocrine resistance, and that sensitivity to endocrine therapy might be reversed by inhibition of this pathway –16. deGrTo the next, studies on the altered expression of β-catenin and mTOR-related molecules were performed to identify a mechanism of action and interaction between β-catenin and mTOR pathways. Cyclin D1 is a well-known regulator of cell-cycle progression, and a target gene of the ERα, β-catenin, and mTOR , 17, 18.Several recent studies reported a relationship between PI3K/Akt/mTOR and β-catenin signaling –23. GrowThe activity of GSK-3β and β-catenin were decreased to the greatest extent by the combination of ICG-001 and rapamycin. The exact mechanisms of interaction between PI3K/Akt/mTOR and the β-catenin pathway are not yet fully understood, and further study is needed for this.In summary, the results of this study suggest that the β-catenin pathway plays a considerable role in breast cancer with acquired tamoxifen-resistance. It is expected that the inhibition of β-catenin will be act as a new targeted therapy in endocrine resistance, such as mTOR inhibitor.


Human T-cell leukemia virus-1 (HTLV-1) is a retrovirus that infects an estimated 15-25 million people worldwide. HTLV-1 is the causative infectious agent of adult T-cell leukemia/lymphoma (ATL) and a neurodegenerative disease (HAM/TSP). While the probability of presenting any symptoms related to HTLV-1 infection is relatively low , the disease progression and prognosis of those infected individuals who develop ATL is fatal, with a median survival range of 8-10 months. Unfortunately, ATL is highly chemotherapy resistant and while many current therapies improve ATL patient survival, the patients consistently relapse. Therefore, a need exists to develop treatments that improve ATL outcome. We have recently identified PRMT5 (protein arginine methyltransferase 5) as a potential target to modulate HTLV-1 gene expression. We find that PRMT5 levels are elevated in T-cell leukemia/lymphoma cell lines compared to freshly isolated naïve T-cells. Likewise, PRMT5 levels are elevated in total PBMCs isolated from ATL patients. However, PRMT5 RNA levels do not correlate to PRMT5 protein levels, suggesting a possible post-transcriptional method of regulation. Furthermore, we also show that PRMT5 levels are elevated during T-cell transformation by using HTLV-1 short-term immortalization assays. Utilizing shRNA vectors, we demonstrate that knockdown of endogenous PRMT5 results in decreased cellular proliferation and increased HTLV-1 viral gene expression. In addition, we observe a decrease in cell proliferation and an increase in viral gene expression when HTLV-1-infected/-transformed cells are treated with a novel small molecule inhibitor of PRMT5 (PRMT5i). Conversely, PRMT5i does not induce re-activation from HIV-1 chronically infected cells. We previously reported a protein-protein interaction between the HTLV-1 accessory protein, p30, and PRMT5. We further show PRMT5 enhances the ability of p30 to inhibit viral gene expression. In conclusion, we find PRMT5 to be a negative regulator of HTLV-1 and a potential target in HTLV-1-mediated disease.


Mrhl RNA negatively regulates canonical Wnt pathway and gets down regulated upon Wnt signaling activation in mouse spermatogonial cells. Also, mrhl RNA regulates expression of genes pertaining to Wnt pathway and spermatogenesis by binding to chromatin. In the present study, we delineate the detailed molecular mechanism of Wnt signaling induced mrhl RNA down regulation in mouse spermatogonial cells. Mrhl RNA has an independent transcription unit and our various experiments like Chromatin Immunoprecipitation (in cell line as well as mouse testis) and shRNA mediated down regulation convincingly show that β-catenin and TCF4, which are the key effector proteins of the Wnt signaling pathway are required for down regulation of mrhl RNA. We have identified Ctbp1 as the co-repressor and its occupancy on mrhl RNA promoter depends on both β-catenin and TCF4. Upon Wnt signaling activation, Ctbp1 mediated histone repression marks increase at the mrhl RNA promoter. We also demonstrate that Wnt signaling induced mrhl RNA down regulation results in an up regulation of various meiotic differentiation marker genes.Long non coding RNAs (lncRNAs) have emerged as important regulators of various biological processes. LncRNAs also behave as response elements or targets of signaling pathway(s) mediating cellular function. Wnt signaling is important in regulating mammalian spermatogenesis.  Air and Kcnq1ot1) (Xist and roX)  (Evx1as and Hoxb5/6as) (Neat1) (1/2-sbs RNAs) (Gas5)  (Evi2 and Lethe)  are becoming increasingly important in cellular regulation. Long non-coding RNAs (lncRNAs) are a subclass of non-coding RNAs, generally greater than 200bp in length and could be sense, antisense, intronic or intergenic . LncRNAscnq1ot1) , dosage and roX) ,5, cell r, etc.) , pluripoxb5/6as) , nuclear (Neat1) , etc. Si (Neat1) , mRNA debs RNAs) , RNA dec) (Gas5) , facilit HOTTIP) ,13, regud Lethe) ,15, regud Lethe)  etc. In d Lethe)  suggestid Lethe) .Mrhl lncRNA, discovered in our laboratory, is encoded in a meiotic recombination hot spot locus of the mouse chromosome 8, in the 15th intron of the phkb gene and transcribed by RNA Polymerase II  activated Wnt signaling suggesting that mrhl RNA acts as a negative regulator of Wnt signaling in spermatogonial cells  and HEK293  were maintained as described previously . Mouse LThe list of antibodies used in the present study is given below with their manufacturer and catalogue number in brackets: β-catenin , TCF4 , Phospho S9 Gsk3β , Phospho S33/S37/T41 , Histone H3 , Alpha tubulin , GAPDH , Ctbp1 , Chd8 , G9a , p300 , Hdac1 , Hdac2 , H3K9ac , H3K9me3 , H3K14ac , RFP , Lamin B , Scp3 .The list of plasmids from Addgene with their plasmid ID and name of the depositor is given in brackets: dnTCF4 , pcDNA3-β-catenin , β-catenin shRNA , scrambled shRNA . Following plasmids were obtained from Sigma–Aldrich and their catalogue number is given in brackets: TCF4 shRNA (TRCN0000012094), Ctbp1 shRNA (TRCN0000085774), TCF4 shRNA targeting 3′ UTR (TRCN0000012093), β-catenin shRNA targeting 3′ UTR (TRCN0000012688), Ctbp1 shRNA targeting 3′ UTR (TRCN0000085773). Following plasmids were generous gifts from Prof. Sanjeev Galande : RFP tagged constructs of full length β-catenin (β-cat FL-RFP), N terminal β-catenin (β-cat N-RFP), C- terminal β-catenin (β-cat C-RFP), GST tagged full-length β-catenin (β-cat FL-GST), Flag tagged TCF4 construct (Flag-TCF4) and Flag tagged Ctbp1 plasmid construct (Flag-Ctbp1) were kind gifts from Prof. G. Chinnadurai . Plasmids were used for transient transfection in Gc1-Spg cells at a final concentration of 1.5 μg/ml using Lipofectamine 2000 as per manufacturer's instructions.All chemical reagents were of AR grade and were purchased from Sigma. Protein A agarose beads (15918-014) and Lipofectamine 2000 (11668027) were purchased from Invitrogen. DNAseI (MO303), NheI (R0131S), BamHI (R0136S) and XhoI (R0146S) were purchased from New England Biolabs. Luciferase assay Kit was supplied by Promega, Streptavidin HRP (ab 7403) obtained from Abcam and Recombinant TCF4 protein from Origene (TP760702)6 cells) were seeded in 90mm culture dishes containing DMEM with 10% FBS. After 48 h, the cells were cultured with fresh medium and incubated for 1 more day. The medium was then collected, centrifuged at 500 × g for 5min, filtered through a 0.2-μm syringe filter and subsequently stored at −20°C till further use. For treatment of Gc1-Spg cells, the control as well as Wnt3a conditioned medium were diluted 2:1 with DMEM containing 10%FBS.L-control or L-Wnt3a cells  was mutated from ATCAAAG to ATCAGCG using Stratagene QuikChange Site-Directed Mutagenesis Kit . For Luciferase assay, Gc1-Spg cells were transfected with 1 μg of the pGL3-Basic vector or 1 μg of mrhl RNA promoter clone in a six-well plate using Lipofectamine 2000. Transfection with CMV-βGal (0.4 μg) plasmid was done as an internal control for transfection efficiency. After 24 h, cells were harvested and processed for Luciferase assay as per the manufacturer's protocol (Promega). Luciferase readings for the pGL3- Basic as well as mrhl RNA promoter plasmid were normalized with the Luminometer readings obtained for CMV-βGal.The 2.4 kb 2, 2% NP40, salmon sperm DNA and 1× binding buffer . The reaction mixture was incubated at room temperature for 1 h. Samples were resolved in 5% native polyacrylamide gel with 0.5× TBE as running buffer. Transfer was done in 0.5× TBE at 380 mA for 45 min followed by which the Nylon membrane was exposed to UV light for 10 min. Then the blot was subjected to 40 min of blocking (3% BSA in 1× TBS) followed by 40 min in streptavidin conjugated HRP (1:1000 dilution in 1% BSA in 1× TBS). After two washes of 10 min each in 1× TBS the blot was analyzed.Equimolar amounts of biotinylated complementary oligos were annealed at 95°C for 5 min. Binding was set for both wild type and mutant oligonucleotides with 2 μg and 4 μg of TCF4 recombinant protein in buffer containing 10% glycerol, 5 mM MgClIn order to obtain the pool of different stages of spermatogenic cells, we selected 7, 14, 16, 18 and 20 days old mice, testes were dissected out, chopped in DMEM medium and filtered through cheese cloth. Cells were fixed in 4% PFA for 15 min at room temperature with gentle agitation. Later, cells were washed once with PBS, permeabilized with 0.5% Triton X-100 in PBS for 10 min with gentle agitation and again washed with PBS at room temperature. Cells were suspended in appropriate amount of PBS, spread over the cover slip and were allowed to dry in humidified chamber for 2–3 h.mrhl RNA  for 1 h with gentle agitation. To remove nonspecific binding of the probe, cells were washed with washing buffer-I  three times for 5 min each at room temperature. Subsequently, cells were washed once for 5 min each with washing buffer-II (2× SSC), washing buffer-III (1× SSC), PBS and processed for immunofluorescence staining. Cells were blocked in 1% BSA for 45 min at 4°C and subsequently incubated at room temperature with primary antibody  for 30 min. Cells were washed three times with PBS for 5 min each, followed by incubation with appropriate Alexa Fluor conjugated secondary antibody (1:400 diluted with 0.1% BSA) for 30 min at room temperature. The cells were washed thrice for 5 min each with PBS and nuclear stained with 1 μg/ml 4′,6′-diamidino-2-phenylindole (DAPI) and mounted in 60% glycerol. The images were acquired in an LSM 10 Meta Confocal microscope (Carl Zeiss) and analyzed by the software provided by Carl Zeiss and intensity quantification was done using ImageJ software.For RNA-FISH experiment, we used Cy5 labeled probe  against mrhl RNA  and hybr  of Gsk3β, decrease in Ser33/Ser37/Thr41 phosphorylated form of β-catenin, nuclear translocation of β-catenin and activation of Sox17 and CyclinD1 gene expression . Activation of Wnt signaling was observed at 18 h of Wnt3a CM treatment as monitored by the nuclear translocation of β-catenin  at −175 bp position. Also, by bioinformatics analysis using TFM explorer we could find putative binding sites for many transcription factors like FoxA1, FoxA2, FoxQ1 and Nkx3-2. In order to validate whether the 1 kb upstream region can function as a promoter element, we cloned the 1kb genomic DNA sequence upstream of mrhl RNA gene in the pGL3 Basic vector and checked for the Luciferase activity of the DNA fragment in Gc1-Spg cells. Luciferase assay showed ∼35-fold increase in Luciferase activity compared to empty vector control, thus demonstrating the promoter activity of the 1 kb upstream DNA fragment  which predominantly contain spermatocytes and represent Wnt activated state as opposed to 7-day-old mouse testis (P7 testis) which predominantly contain spermatogonia and represent Wnt repressed state  at −175 position [further referred as Mrhl(−175 bp)]. TCF4 is a key transcription factor of Wnt signaling and its regulatory function is dependent on β-catenin. The β-catenin–TCF4 complex occupies the Wnt target gene promoters and brings about activation/repression based on the recruitment of either co-activators or co-repressors  experiments using anti β-catenin and anti TCF4 antibodies in control medium treated and Wnt3a CM treated Gc1-Spg cells as well as in P7 and P21 mice testes. The data presented show the occupancy of TCF4  only upon Wnt signaling activation (Wnt3a-CM treated Gc1-Spg and P21 testis chromatin) but not in control medium treated Gc1-Spg or P7 testis chromatin. A 250 bp upstream region of β-actin promoter [β-actin (−250 bp)] and also the region from −800 bp to −1 kb within the mrhl RNA gene promoter [Mrhl (−1 kb)] which does not contain a TCF4 binding site were used as negative controls. As a positive control, we used the 300 bp upstream region of CyclinD1 promoter [Ccnd1(−300 bp)] which contains a TCF4 binding site. The occupancy of TCF4 at Ccnd1(−300 bp) even in the absence of Wnt signaling activation (control medium treated Gc1-Spg and P7 testis) is consistent with the earlier reports demonstrating occupancy of TCF4 at subset of its target gene promoters even in the absence of Wnt signaling activation  in Gc1-Spg cells. dnTCF4 is a truncated form of TCF4 with N-terminal deletion of the first 32 amino acids which renders TCF4 incapable of interacting with β-catenin . This shows that physical interaction of β-catenin and TCF4 is required for the down regulation of mrhl RNA expression upon Wnt signaling activation.Occupancy of β-catenin and TCF4 at the Wnt responsive elements (WRE) of -catenin . After 2mrhl RNA expression, but cells treated with either β-catenin or TCF4 shRNA followed by Wnt3a CM treatment failed to down regulate mrhl RNA expression (expression comparable to control medium treated cells). Luciferase reporter assay with the mrhl RNA promoter construct also showed significantly reduced reporter activity upon Wnt3a CM treatment while there was no reduction in the Luciferase activity upon treatment with β-catenin or TCF4 shRNA . In order to confirm the specificity of shRNA mediated knockdown experiments, we carried out the rescue of β-catenin and TCF4 expression. For this purpose we used shRNAs targeting the 3′ UTR of β-catenin and TCF4 mRNAs and carried out over expression of β-catenin and TCF4 protein using plasmid constructs containing the cDNA of β-catenin and TCF4. As seen in Figure mrhl RNA was not down regulated (in control medium as well as Wnt3a CM) upon β-catenin or TCF4 shRNA treatment . Further we examined whether the N-terminal or the C-terminal domain of β-catenin is required for the down regulation of mrhl RNA. We used RFP tagged constructs of full length β-catenin (β-cat FL-RFP), N terminal β-catenin , C-terminal β-catenin . Upon knock down of β-catenin using the 3′ UTR targeting shRNA, we over expressed β-cat FL-RFP, β-cat N-RFP and β-cat C-RFP . Towards this objective, we sought to examine the co-occupancy of Ctbp1 and β-catenin/TCF4 as well as Chd8 and β-catenin/TCF4 on the mrhl RNA promoter by ChIP and sequential ChIP (ChIP-Re ChIP) experiments. We observed that Ctbp1 occupied the mrhl RNA promoter element at Mrhl(−175 bp)  we carried out sequential ChIP experiments. It is now very well established that if two proteins co-occupy at a particular genomic site, in a quantitative context the fold enrichment (over input DNA) of the sequential ChIP is equal to the product of the fold enrichments of the individual ChIP (Mrhl(−175 bp)   was seen only under the conditioned Wnt signaling activation (Wnt3a CM treated Gc1-Spg cells) and the P21 testicular chromatin.In addition to the recruitment of β-catenin at the TCF4 binding site within the promoters of Wnt target genes, the molecular mechanism envisages the recruitment of either co-activators or co-repressors at these sites . Recent ) Figure  while Ch) Figure . To deteual ChIP . Our seq) Figure  and D as) Figure  and F. Tmrhl RNA, we went ahead with silencing the expression of Ctbp1 in Gc1-Spg cells using the shRNA approach. Ctbp1 protein levels were drastically reduced upon Ctbp1 shRNA treatment in Gc1-Spg cells treated with control medium as well as Wnt3a CM   was regulated by β-catenin and TCF4 since the silencing of β-catenin and TCF4 led to loss of Ctbp1 occupancy at Mrhl(−175 bp) in Wnt3a CM treated Gc1-Spg cells  proximal promoter region only upon Wnt signaling activation while p300 occupied Mrhl(−175 bp) even in the absence of Wnt signaling activation  upon Ctbp1 shRNA treatment .Since only Ctbp1 but not Chd8 occupied the promoter of M Figure . Analysi) Figure . The occs Figure . It is ks Figure ,36. Few n Figure . In a wat Figure . Howevers Figure . Similars Figure . Therefomrhl RNA promoter and also correlated them with the occupancy of Ctbp1 on the mrhl RNA promoter. We found that H3K9 acetylation  upon Wnt signaling activation (Wnt3a CM treated Gc1-Spg and P21 testis). In a control set of experiments, no such significant changes were observed at the Mrhl (−1 kb) promoter region for any of these histone modification marks. These changes in the histone modifications at Mrhl(−175 bp) correlated very well with the observation that Ctbp1 occupied Mrhl(−175 bp) only upon Wnt signaling activation.Previous studies have uncovered the mechanism of Ctbp1 mediated transcriptional repression ,36. As mn Figure  and H3K1n Figure  showed an Figure  showed ain vivo differentiate into meiotic spermatocytes in the mammalian testis. It is not clear at present which factors govern this commitment to differentiation in vivo. Based on the fact that Wnt signaling is activated in meiotic spermatocytes and our observation that mrhl RNA is down regulated in spermatocytes, we were curious to examine the Wnt3a ligand treated Gc1-Spg cells particularly with respect to the expression of differentiation markers. For this purpose, we assessed the transcript levels of few premeiotic markers  and meiotic markers . Expression of c-Kit is increased in spermatogonia committed to meiotic prophase  that there is no significant perturbation of expression of the differentiation markers under those conditions . Therefore from this experiment we infer that the down regulation of mrhl RNA in the context of Wnt signaling activation is essential for regulating the expression of meiotic differentiation markers. At this juncture, we were curious to examine whether such an inverse relationship between the down regulation of mrhl RNA expression and β-catenin appearance in the nucleus  does reflect the in vivo situation. For this purpose, we performed RNA-FISH (RNA-fluorescent in situ hybridization) using LNA (locked nucleic acid) probes on the meiotic spreads prepared from mice testes to examine the expression and localisation of mrhl RNA at different meiotic prophase stages. SCP3  localization pattern along the chromosomes was used to identify cells of different stages of meiotic prophase namely leptotene, zygotene/pachytene and pachytene in the meiotic spreads while β-catenin localization was used to assess the Wnt signaling activation. As can be seen from Figure mrhl RNA. This observation agrees with the recent report of Kerr et al. demonstrating the nuclear localisation of β-catenin in pachytene spermatocytes . Day 7 (P7) mouse testis contains spermatogonia as the predominant germ cells while at day 14 (P14) early spermatocytes are present  cells. As per our knowledge, this is the first report of a lncRNA being regulated by Wnt signaling pathway. Although there are few reports of lncRNA as targets of Wnt signaling, the detailed molecular mechanism(s) involved in their regulation has not been studied. The present study describes the molecular events leading to down regulation of mrhl RNA upon Wnt signaling in mouse spermatogonial cells.Mammalian spermatogenesis is a unique process of cellular differentiation encompassing three different phases namely (i) mitotic phase, generating more of spermatogonial cells, (ii) meiotic phase, generating genetically diverse haploid spermatids and (iii) spermiogenesis, involving maturation of spermatids to sperms that includes nuclear reshaping and chromatin packaging ,49. Wnt al cells . In thisMrhl RNA is an intronic lncRNA transcribed from the 15th intron of mouse phkb gene as an independent transcription unit and the proximal promoter region contains many putative transcription factor binding sites including that of transcription factor TCF4. The mechanism of mrhl RNA down regulation is executed mainly at this TCF4 binding site. The requirement of beta catenin along with TCF4 for the down regulation of mrhl RNA only upon activation of Wnt signaling further qualifies mrhl as a Wnt target gene. Further, the requirement of a co-repressor in form of Ctbp1 accompanied by a repressive histone modification signature (reduced H3K9ac and H3K14ac and increased H3K9me3) at the mrhl RNA proximal promoter fits in nicely with the very well documented modes of regulation of gene expression as a consequence of Wnt signaling activation. Ctbp1 has been shown by Shi et al. to be a part of a multimeric complex which consists of proteins with multiple histone modifying activities and propose a repressive mode of action for the Ctbp complex which is coordinated by the histone modifications catalyzed by Hdac1/2 and G9a   which is a RNA polymerase II transcribed gene employs similar mechanism for regulation as has been established for many of the other protein coding genes. It is pertinent to point out here that Wnt signaling although brings about down regulation of the mrhl gene; the transcription of phkb gene is not perturbed. It would be interesting to study in future how the transcription is differentially regulated at a parent gene and a gene encoded within an intron of the parent gene. and G9a . Ctbp1 with TCF4 . But, inith TCF4 . Similarith TCF4 . The repha (ERα) . In theiin vivo correlate for our experiments performed in a cell culture system. In our recent report, where we have studied the role of mrhl RNA in the regulation of gene expression at the level of chromatin we showed a perfect correlation of cell culture system and mouse testicular tissue, proving the biological relevance of mrhl RNA mediated gene regulation  as compared to P7 testis . In this context, an inverse correlation between the expression status of mrhl RNA and Wnt signaling raises an important biological question as to whether Wnt signaling regulates the meiotic commitment and differentiation of B type spermatogonia into meiotic spermatocytes and what is the role of mrhl RNA if any in this differentiation process. In these experiments we also observed that the β-catenin staining of zygotene/pachytene spermatocytes (Figure mrhl RNA, in trans, abrogated the up regulation of these meiotic differentiation markers, showing the necessity of Wnt signaling induced mrhl RNA down regulation for the differentiation. A model summarizing our findings reported in the present study has been depicted in Figure mrhl RNA alone is not sufficient for up regulation of meiotic markers expression as revealed from our microarray data. Hence, we would like to believe that Wnt signaling triggers several cascading events in order to initiate meiotic commitment and in this process down regulation of mrhl RNA expression is a critical event. Thus a detailed analysis of signaling events leading to commitment of spermatogonia towards differentiation into meiotic prophase spermatocytes would throw much light in our understanding of this very important biological phenomenon and more particularly, the role of mrhl RNA in this process. It is also worth mentioning here that the role of mrhl RNA in meiotic commitment of spermatogonial cells may be only one of its several biological roles, since the down regulation of mrhl RNA alters several other genes not associated with Wnt signaling pathway and also occupies several chromatin loci that are not connected with Wnt signaling (In most of our experiments, we have also used mouse testicular tissue chromatin as an gulation . Mrhl RNs Figure  was moreignaling .


Wnt/β-catenin signaling has been suggested to regulate proximal-distal determination of embryonic lung epithelium based upon genetically modified mouse models. The previously identified and characterized small molecule inhibitor IQ1 can pharmacologically decrease the interaction between β-catenin and its transcriptional coactivator p300, thereby enhancing the β-catenin/CBP interaction. Inhibition of the β-catenin/p300 interaction by IQ1 blocks the differentiation of embryonic stem cells and epicardial progenitor cells; however, whether differential coactivator usage by β-catenin plays a role in proximal-distal determination of lung epithelium is unknown.We examined the effects of inhibiting the β-catenin/p300 interaction with IQ1 on lung branching morphogenesis in mouse embryos in utero and mouse embryonic lung organ culture ex vivo. The phenotype of IQ1 treated lungs was analyzed by epithelial staining, histology, quantitative PCR and in situ hybridization.Inhibition of the β-catenin/p300 interaction by IQ1 disrupted the distal branching of mouse lung epithelium both in utero and ex vivo. IQ1 proximalized lung epithelium with decreased expression of the genes Bmp4 and Fgf10, hallmarks of distal lung determination, and increased expression of the proximal genes Sox2 and Scgb1a1 (CC10) as shown by quantitative PCR and in situ hybridization. The disruption of branching was reversible ex vivo as branching was reinitiated after removal of IQ1 from the media.The results demonstrate that the β-catenin/p300 interaction plays a critical role in proximal-distal determination of the epithelium in mouse lung branching morphogenesis and β-catenin/p300 inhibition pharmacologically proximalizes lung epithelium. The ability to regulate proliferation and differentiation of the lung has important consequences in regards to both premature birth and bronchopulmonary dysplasia  and dise21H22N4O2, molecular weight 362.42) and ICG-001  are specific inhibitors of the interaction between β-catenin and the transcriptional coactivators p300 and CBP respectively. IQ1 inhibits the interaction between β-catenin and p300 indirectly via targeting the PR72/130 subunit of PP2A and thereby blocking PP2A/Nkd complex formation [Chemical genetic approaches offer a complementary method to genetic manipulation, to selectively modulate the transcriptional role of β-catenin. To activate transcription, β-catenin must translocate to the nucleus to interact with transcription factors e.g. members of the TCF/LEF in canonical Wnt signaling as well as various transcriptional co-activators. We have identified and characterized a number of small molecules that specifically inhibit the protein-protein interaction between β-catenin and either of its two Kat3 coactivators i.e. Creb binding protein (CBP) or p300. Specifically inhibiting either the β-catenin/CBP or the β-catenin/p300 interaction does not disturb other protein-protein interactions involving β-catenin, including the β-catenin/E-Cadherin interaction that is critically responsible for epithelial cell-cell adhesion. Therefore, specific well-characterized small molecule pharmacologic tools provide an advantage for loss-of-function analysis of multifunctional proteins like β-catenin. An additional potential advantage of utilizing a pharmacologic approach is the ability to temporally utilize small molecule pharmacologic agents to manipulate cell proliferation and differentiation for the purpose of regenerative medicine. IQ1  or ICG-001 (220 mg/kg/day) mixed in peanut butter in addition to their regular diet. The animal study protocol was approved by the IACUC at the University of Southern California.TOPGAL mice were obtained from The Jackson Laboratory. Vaginal plug at noon was considered as day 0.5 of pregnancy (E0.5). For administration of IQ1 or ICG-001 to embryos Lung samples were fixed in 4% paraformaldehyde for 1 hour to overnight and washed in phosphate buffered saline (PBS). The lungs were incubated in a solution of NBT/BCIP Ready-to-Use Tablets (Roche Applied Science) according to the manufacturer’s directions. After development of color, excess staining was removed by dehydration and rehydration through a series of methanol washes. The stained lungs were cleared in graded steps with glycerol before photographing. Branching tips of the stained lungs were counted under a bright-field stereomicroscope. For histological analysis, the stained lungs were re-fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and counterstained with Nuclear Fast Red (Electron Microscopy Sciences). For β-galactosidase staining, lungs were dissected, fixed in 0.5% glutaraldehyde with 2 mM of magnesium chloride and stained as described previously. The images were captured using an AxioImager Z1 and AxioVision digital image processing software (Carl Zeiss) or SZX7 stereomicroscope and infinity2 imaging system (Olympus).Lungs were dissected from mouse embryos in cold sterile Hanks’ Balanced Salt Solution under a stereomicroscope. The lungs were randomized by size and obvious outliers were excluded. The lungs were placed on a polycarbonate membrane filter (Whatman) floating on 1 ml of serum-free BGJb medium (Invitrogen) with penicillin/streptomycin in 60 mm Center-well Organ Culture Dishes . The lung explants were positioned at the gas–liquid interface on the membrane filters. The Cultures were kept at 37°C in 5% CO2 and media was changed daily.Bmp4 , mouse Fgf10 , mouse Nkx2-1 , mouse Shh , mouse Scgb1a1 . Melt curve analysis and gel electrophoresis were used to examine the specificity of amplified products. Data were normalized to the reference gene, mouse Gapdh. Relative expression levels were calculated using the 2^-ddCt method. Data are presented as mean ± SD. Differences in means between two experimental groups were analyzed using two-sample, two-tailed Student’s t-test. p < 0.05 was considered significant.The lung explants were homogenized with TRIzol® reagent (Invitrogen) by passing through a 25-gage needle and total RNA was isolated according to the manufacturer’s instructions. The concentration of total RNA was measured using a NanoDrop® spectrophotometer and reverse transcription was performed with an iScript cDNA Synthesis Kit (Bio-Rad). Quantitative PCR amplification was performed using SYBR Green PCR master mix reagent (BioRad) and a MyiQ thermal cycler (BioRad) with the following gene-specific primers: mouse in situ hybridization as previously described. Digoxigenin-labeled anti-sense RNA probes were synthesized from subcloned mouse gene templates using DIG RNA Labeling Kit (Roche Applied Science). Hybridization was colorized with ALP conjugated anti-DIG antibody (Roche Applied Science) and NBT/BCIP Ready-to-Use Tablets (Roche Applied Science). Experiments were independently repeated at least three times. The plasmids including cDNA of mouse Nkx2-1 and Bmp4 were generously provided by Dr. Changgong Li.The lung explants were fixed in 4% paraformaldehyde for 4 hours to overnight, stored in 100% methanol at −20°C, bleached in 3% hydrogen peroxide in methanol, rehydrated and subsequently underwent Lung samples were fixed in 4% paraformaldehyde overnight, embedded in OCT compound or paraffin and sectioned. For immunohistochemistry, the sections were blocked in 1% bovine serum albumin (BSA) (Jackson ImmunoResearch) and incubated with primary antibodies. The antibodies used were FITC mouse anti-β-catenin  and anti-α-SMA . For whole mount PECAM-1 staining, fixed lungs were dehydrated in methanol, bleached in 3% hydrogen peroxide in in methanol, rehydrated, blocked in 1% BSA, incubated with anti-PECAM-1 antibody  overnight at 4°C, washed in PBS, incubated with HRP-conjugated anti-goat IgG (Santa Cruz) and colorized with diaminobenzidine (DAB) substrate (Sigma).Wnt signaling apparently plays a critical role during lung branching morphogenesis. We found that murine embryonic lung expresses endogenous alkaline phosphatase (ALP) activity in the epithelium. The epithelium can be selectively stained utilizing the endogenous ALP activity with the chromogenic substrate, nitro-blue tetrazolium chloride and 5-bromo-4-chloro-3′-indolyphosphate p-toluidine salt (NBT/BCIP). We therefore sought to utilize NBT/BCIP to visualize epithelial branching morphogenesis in whole mount mouse lung. In the event, we stained mouse lung at four different embryonic stages from E11.5 to E14.5 with NBT/BCIP. We observed blue-purple staining specifically visualizing lung epithelial structures during branching morphogenesis at all four time points  as distal marker genes and Sox2 and Scgb1a1 (CC10) as proximal marker genes. The critical growth factors involved in branching morphogenesis  were examined at 6 hours and 24 h. As anticipated, IQ1 significantly decreased the expression of the distal genes, Bmp4, Fgf10 and Nkx2-1 . We selected ex vivo.The proximalization resulting from IQ1 inhibition of the β-catenin/p300 interaction phenotypically resembles those seen utilizing genetic deletion of β-catenin ,14. Therβ-catenin is a multifunctional protein that beyond its role as a transcriptional activator of Wnt signaling, is also an important protein component of adherens junction, which are important for epithelial cell-cell adhesion. To test if the inhibition of the β-catenin/p300 interaction affected the expression and localization of β-catenin in the lung epithelium, we next examined β-catenin by immunohistochemistry. We found β-catenin widely expressed in both the epithelium and mesenchyme of the control treated lungs  and Fgf10 and increased expression of the proximal genes and Sox2 and Scgb1a1 (CC10)  has proven extremely safe in both Investigational New Drug (IND) enabling toxicology studies and in a Phase Ia clinical trial [ex vivo small molecule modulation of stem and progenitors cells offers another avenue to pursue. In that regard, Banerjee et al. demonstrated that human embryonic stem cells (hES cells) directed to alveolar type II (ATII) lung progenitor cells, could be differentiated into an alveolar type I (ATI) phenotype following incubation with the CBP/β-catenin antagonist ICG-001 [ex vivo the lung development process [Activation of the Wnt/β-catenin signaling cascade is critical during lung development and has been implicated in the restoration of normal tissue structure and function, as well as remodeling/fibrosis in a number of organs, including the lung, suggesting that this developmental pathway can be reactivated in adult tissues following injury ,27. Howeal trial . In rega ICG-001 . One wee process . Small mOur study provides strong evidence that the interaction between β-catenin and p300 is critically important in proximal-distal axis determination during mouse lung branching morphogenesis. Our results are consistent and add further refinement to multiple previous reports regarding the role of β-catenin on proximal-distal determination in the lung based upon genetic deletion of β-catenin as well as previous reports on the differential roles of the coactivators CBP and p300 during lung development. The chemical genomic approach utilized herein further elucidated and clarified the specific role of the coactivator p300 in the regulation of Wnt/β-catenin driven branching morphogenesis in the lung. This information, along with the ability to safely pharmacologically manipulate differential coactivator usage in the Wnt/β-catenin cascade provides multiple therapeutic opportunities to investigate going forward.