Therefore, the present study aimed to investigate the potential role of H2S in atherosclerosis and the underlying mechanism with respect to chemokines  and chemokine receptors  in macrophages. Mouse macrophage cell line RAW 264.7 or mouse peritoneal macrophages were pre-incubated with saline or NaHS , an H2S donor, and then stimulated with interferon-γ (IFN-γ) or lipopolysaccharide (LPS). It was found that NaHS dose-dependently inhibited IFN-γ or LPS-induced CX3CR1 and CX3CL1 expression, as well as CX3CR1-mediated chemotaxis in macrophages. Overexpression of cystathionine γ-lyase (CSE), an enzyme that catalyzes H2S biosynthesis resulted in a significant reduction in CX3CR1 and CX3CL1 expression as well as CX3CR1-mediated chemotaxis in stimulated macrophages. The inhibitory effect of H2S on CX3CR1 and CX3CL1 expression was mediated by modulation of proliferators-activated receptor-γ (PPAR-γ) and NF-κB pathway. Furthermore, male apoE−/− mice were fed a high-fat diet and then randomly given NaHS  or DL-propargylglycine . NaHS significantly inhibited aortic CX3CR1 and CX3CL1 expression and impeded aortic plaque development. NaHS had a better anti-atherogenic benefit when it was applied at the early stage of atherosclerosis. However, inhibition of H2S formation by PAG increased aortic CX3CR1 and CX3CL1 expression and exacerbated the extent of atherosclerosis. In addition, H2S had minimal effect on the expression of CCL2, CCL5, CCR2 and CCR5 in vitro and in vivo. In conclusion, these data indicate that H2S hampers the progression of atherosclerosis in fat-fed apoE−/− mice and downregulates CX3CR1 and CX3CL1 expression on macrophages and in lesion plaques.Hydrogen sulfide, as a novel gaseous mediator, has been suggested to play a key role in atherogenesis. However, the precise mechanisms by which H It has become clear that H2S fulfills a wide range of physiological functions and plays important roles in the pathogenesis of various cardiovascular diseases, such as hypertension, pulmonary hypertension, and myocardial injury 2S has shed light on the potential role of the gas in atherosclerosis. Wang et al. first reported a direct correlation between endogenous H2S and atherosclerosis in apoE−/− mice 2S may hinder the development of atherosclerosis by inhibiting vascular smooth muscle cell proliferation, adhesion molecules expression in endothelial cells and foam cell formation 2S are still far from clear. The present study seeks to investigate whether H2S also reduced the expression of chemokines and their receptors, which have been shown to play a key role in inflammatory conditions and atherosclerotic lesion development Hydrogen sulfide is commonly considered to be a toxic gas with the smell of rotten eggs. However, it is generated endogenously during cysteine metabolism in a reaction catalyzed by two pyridoxal phosphate-dependent enzymes, cystathionine β-synthase  and cystathionine γ-lyase  2S may contribute to a better understanding of the precise mechanisms by which H2S hinders the progression of atherosclerosis.In atherogenesis, chemokines and chemokine receptors may coordinate communication between inflammatory cellular components of the peripheral blood and cellular components of the arterial wall, thereby regulating leukocyte influx, capture, efflux and activation, as well as proliferation and/or apoptosis of reside cells in the plaque 2S could regulate the expression of chemokines  and chemokine receptors  in vivo and in vitro. We found that H2S inhibited CX3CR1 expression on stimulated macrophages and reduced aortic CX3CR1 expression in fat-fed apoE−/− mice. The reduction in CX3CR1 expression induced by H2S was mediated by peroxisome proliferators-activated receptor-γ pathway. Moreover, H2S downregulated CX3CL1 expression in stimulated macrophages and decreased aortic CX3CL1 expression in vivo by suppressing the activation of nuclear factor (NF-κB). However, H2S had negligible effect on CCL2-CCR2 axis and CCL5-CCR5 axis in vitro and in vivo. Our findings suggest that H2S protects against the formation of atherosclerotic lesions in mice, along with downregulating macrophage CX3CR1 and CX3CL1 expression by a PPAR-γ and NF-κB dependent mechanism.Accordingly, in the present study, we examined whether HUsing RT-PCR and western blot analysis, we found that NaHS dose-dependently decreased the mRNA and protein expression of CX3CR1 in RAW264.7 cells stimulated with either IFN-γ (500 U/ml) or LPS (10 µg/ml) . Similar2S-induced downregulation of CX3CR1 in stimulated macrophages. It was found that unstimulated RAW264.7 cells exhibited a very low level of DNA binding activity for PPAR-γ, whereas a relatively high DNA binding activity for PPAR-γ was observed in mouse peritoneal macrophages (The nuclear receptor PPAR-γ has the inherent capacity to regulate expression of macrophage chemokine receptors (CX3CR1 and CCR2) in lipid-mediated inflammation in atherosclerosis lesions rophages . This isrophages . Regardlrophages .2S on CX3CR1 expression in stimulated macrophages, GW9662, a selective PPAR-γ antagonist, was utilized. As shown in 2S may inhibit CX3CR1 expression and CX3CR1-mediated chemotaxis in stimulated macrophages in a PPAR-γ-dependent mechanism.To further test whether PPAR-γ signaling was necessary for the inhibitory effect of H2S synthesis activity assay . 24 weeks after fat feeding, mice were sacrificed and the extent of atherosclerotic lesions was assessed by ultrasound biomicroscopy (UBM) and histological analysis. UBM assessment showed that early NaHS treatment significantly impeded plaque development in the aorta, as characterized by a significant decrease in the brachiocephalic artery (BCA) plaque size, common carotid artery (CCA) intima-media complex thickness (IMT) and the aortic arch IMT . As shown in Moreover, administration with PAG significantly depleted endogenous HIn addition, treatment with NaHS or PAG did not affect body weight or the concentrations of plasma lipids but resulted in a slight reduction in blood pressure  and S6.2S may directly act on plaque macrophages to lower CX3CR1 expression in vivo  secretion in microglia, a specialized form of macrophages residing in central nervous systems 2S stimulated the activation of human monocytes with the generation of cytokines 2S on macrophage recruitment and hemostasis in atherosclerotic lesions has not been determined. Recently, chemokines  and chemokine receptors  have been strongly implicated to play critical roles in mediating inflammation, macrophage recruitment and atherosclerosis progression by multiple lines of evidence, including expression, functional and epidemiologic data 2S in regulating CCL2-CCR2, CCL5-CCR5 and CX3CL1-CX3CL1 expression in RAW264.7 cells and mouse peritoeal macrophages. Activation of macrophages was achieved by classic stimulators, IFN-γ and LPS. Cell activation resulted in enhanced CX3CL1 and CX3CR1 expression and chemotaxis towards CX3CL1. Supplement of exogenous H2S dose-dependently reduced the LPS or IFN-γ induced-upregulation of CX3CL1 and CX3CR1 in macrophages. Similarly, overproduction of endogenous H2S via CSE overexpression also inhibited CX3CL and CX3CR1 expression in stimulated macrophages. As a result, either exogenous applied H2S or overexpression of CSE reduced macrophage migration in response to CX3CL1 after stimulation with IFN-γ or LPS. Collectively, our findings demonstrate that H2S plays a crucial role in regulating CX3CL1 and CX3CR1 expression and CX3CR1-mediated chemotaxis in stimulated macrophages.H2S in regulating CX3CL1 and CX3CR1 expression during atherosclerosis and to establish its pathophysiological relevance in vivo, we examined the effect of H2S on CX3CL1 and CX3CR1 expression in fat-fed apoE−/− mice. Administration of NaHS at either the early or the advanced stage of atherosclerosis suppressed the aortic expression of CX3CL1 and CX3CR1, together with reduced plaque size and IMT thickness in the main branches of the aortic arch. In contrast, inhibition of endogenous H2S formation upregulated aortic expression of CX3CL1 and CX3CR1, thus exacerbating atherosclerosis in aorta. Furthermore, immunofluorescent staining revealed that CX3CR1 colocalized with macrophages, suggesting that the reduction in plaque CX3CR1 expression is predominantly by macrophages and that other cell types are likely to play insignificant roles. These findings indicated that H2S may directly act on plaque macrophages and inhibit CX3CR1 expression in vivo. These results are consistent with the findings of in vitro studies with mouse macrophage cell line or isolated mouse peritoneal macrophages. However, flow cytometry analysis found that injection of NaHS or PAG did not alter CX3CR1 expression on circulating monocytes isolated from fat-fed aopE−/− mice. This lack of change may be due to the short half-life (approximately 6 hours) of circulating monocytes, such that their exposure time to H2S is inadequate to inhibit CX3CR1 expression. This result also supports the concept that H2S induced reduction in CX3CR1 expression occurs in macrophages within plaques but not in the circulating monocytes. A decline in CX3CR1 expression in macrophages at the site of lesions may be beneficial, eg suppressing the recruitment and retention of macrophages in blood vessel walls and thereby retarding the development and progression of atherosclerosis.To further demonstrate the involvement of H2S inhibited CX3CL1 expression in stimulated macrophages and in aorta tissues. However, it had no effect on plasma CX3CL1 level in vivo. It may be attributed to the fact that CX3CL1 is predominantly a membrane-bond chemokine. Furthermore, H2S did not affect CCL2-CCR2 and CCL5-CCR5 expression both in vitro and in vivo. These observations support the notion that CCL2-CCR2, CCL5-CCR5 and CX3CL1-CX3CR1 axis impact the pathogenesis of atherosclerosis independently.The present study found that H2S regulates CX3CR1 expression and CX3CR1-mediated chemotaxis in macrophages with respect to the PPARγ pathway. Here, we showed that H2S-induced inhibition of CX3CR1 expression in stimulated macrophages is dependent on PPAR-γ activation. Support for this concept comes from the observation that NaHS dose-dependently increased the DNA binding activity of PPAR-γ in RAW264.7 cells and mouse peritoneal macrophages stimulated with IFN-γ or LPS. CSE overexpression also enhanced the activation of PPAR-γ in IFN-γ or LPS stimulated RAW264.7 cells. Moreover, our notion regarding the involvement of PPAR-γ was further strengthened by the finding that blockage of PPAR-γ activation by GW9662 markedly reduced the inhibition of CX3CR1 expression induced by NaHS or CSE overexpression in stimulated macrophages. Consequently, GW9662 eliminated exogenous or endogenous H2S-induced inhibition of CX3CR1-mediated chemotaxis in activated macrophages. Together, these results indicate that PPAR-γ is possibly involved in the signaling transduction of H2S-induced inhibition of CX3CR1 expression in macrophages.Recent studies have shown that PPARγ has the inherent capacity to regulate expression of macrophage chemokine receptors CX3CR1 in the lipid-mediated inflammation process of atherosclerosis lesions 2S is a highly reactive molecule that readily reacts with free reactive oxygen species (ROS) and reactive nitrogen species (RNS) 2S may suppress the nitration of PPAR-γ by directly scavenging ROS and/or RNS, thus provoking the activation of PPAR-γ and downregulating the expression of CX3CR1 in macrophages. However, the precise mechanism by which H2S regulates the activation of PPAR-γ in stimulated macrophages is still far from clear. Further investigations are required to address this and to resolve the detailed structural basis for PPAR-γ induction of CX3CR1 expression.H2S. The present study found that H2S inhibited IκB degradation, the key step to NF-κB nuclear translocation and activation, and decreased the DNA binding activity of NF-κB p65 in macrophage nuclear extracts. This suggests that H2S may downregulate CX3CL1 expression in macrophages by suppressing the activation of NF-κB pathway.CX3CL1 expression has been shown to be modulated by NF-κB activation 2S on athoerslcerosis and macrophage CX3CR1-CX3CL1 expression. Further work is needed most likely in CX3CR1/CX3CL1 gene modified mice to probe the exact linkage involved. In addition to its effect on the macrophage CX3CR1-CX3CL1 expression, H2S has been shown to impede the pathogenesis of atherosclerosis in various ways 2S plays a role in regulating vascular smooth muscle cell (VSMC) proliferation and apoptosis. It also inhibits atherogenic modification of low-density lipoprotein (LDL), interferes with vascular calcification and is involved in platelet function.Finally, it is to be noted that our data are still insufficient to establish a clear causal link between the inhibitory effect of H2S formation in the aorta accompanies the development and progression of atherosclerosis. H2S may have an anti-atherogenic benefit and this effect seems to be more potent when it is applied during the early stage of atherosclerosis. H2S may prevent the progression of atherosclerosis, along with downregulating macrophage CX3CR1 and CX3CL1 expression by a PPAR-γ and NF-κB dependent mechanism. Our study therefore provides evidence that H2S donors may be a possible new therapeutic strategy to counteract the development of atherosclerosis or other immune diseases.In summary, the present study suggests that deficiency of endogenous HRAW 267.4 murine macrophage cells were obtained from ATCC. Cells were grown in Dubbelco’s Modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) , 100 units/ml penicillin, and 100 mg/ml streptomycin and plated on 6-well plates or 100 mm tissue culture dishes 24 h before experiments. Near-confluent cultures were switched to medium containing 0.5% FBS for 24 h before NaHS treatment or stimulation with LPS or IFN-γ. No cells older than passage 10 were used, because RAW cells showed highly variable responses at higher passage numbers.Peritoneal macrophages were isolated from 10-week, male C57BL/6J mice. A 3% thioglycolate solution  was injected intraperitoneally and, 4 days later, peritoneal macrophages were harvested through phosphate-buffered saline lavage. Ten milliliters of ice-cold phosphate-buffered saline was injected into the mouse peritoneal cavity and gently removed. Once collected, cells were seeded in RPMI medium 1640 supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin for 4 h and then rinsed and maintained in RPMI medium 1640 supplemented with 0.5% FBS for 24 h before NaHS treatment or stimulation with LPS or IFN-γ.Cells were rinsed twice with serum-free culture medium before treatment and all treatments were carried out in serum-free culture medium containing penicillin and streptomycin. Cells were pre-incubated with saline or NaHS  for 6 hours and then stimulated with IFN-γ (500 U/ml) or LPS (10 µg/ml) for 12 hours in the continuous presence of NaHS. For experiments using inhibitors, cells were pre-incubated with GW9662 (10 µM) for 1 hour before NaHS addition.−/− mice were purchased from the Animal Center of the Beijing University, Beijing, China. At the age of 8 weeks, feeding of animals on a high-fat diet began that contained 21% fat from lard and was supplemented with 0.15% (w/w) cholesterol. At indicated time points , mice were sacrificed by an intraperitoneal (i.p.) injection of a lethal dose of pentobarbitone. To investigate the role of H2S in atherosclerosis, different drug administration schedules were used. In the early-NaHS treatment study, thirty mice began to receive NaHS  4 weeks after fat feeding and were sacrificed 24 weeks after fat feeding. In the delayed-NaHS treatment study, thirty mice began to receive NaHS  12 weeks after fat feeding and were sacrificed 24 weeks after fat feeding. In DL-propargylglycine (PAG) treatment study, eighteen mice began to receive PAG  4 weeks after fat feeding and were sacrificed 12 weeks after fat feeding. Samples of aortas and plasma were harvested and stored at –80°C for subsequent measurement.The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Shanghai JiaoTong University School of Medicine. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Shanghai JiaoTong University School of Medicine (Permit Number: [2011]-23). C57BL/6J male apoESystolic blood pressure (SBP) was measured at the beginning and the end of the studies by using a non-invasive computerized tail cuff system . A series of readings was taken and an average of 3–5 BP readings was used. Blood samples were drawn from the left ventricle of mice at the end of the studies and plasma lipid concentrations were determined in heparinized plasma.TM accession number: NM_145953) from C57BL/6J mouse liver using the primers 5′-CAT GCA GAA GGA CGC CTC TTT GA-3′ and 5′-TTA AGG GTG CGC TGC CTT CAA AGC -3′. The amplified open reading frame of CSE was subcloned into the TA cloning vector . A positive clone containing CSE open reading frame insert was sequenced to confirm the accuracy of the inserted CSE sequence. The constructs containing CSE cDNA were cleaved and subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen). The CSE expression vectors or the control vectors (pcDNA3.1 empty vectors) were transiently transfected into RAW264.7 cells using FuGENE HD Transfection Reagent , according to manufacturer’s instruction.PCR was used to amplify the open reading frame of CSE  according to the manufacturer’s protocol. The concentration of isolated nucleic acids was determined spectrophotometrically by measuring the absorbance at 260 nm and the integrity was verified by ethidium bromide staining of 18 S and 28 S rRNA bands on a denaturing agarose gel. All samples were thereafter stored at −80°C until required. 1 µg of RNA was reversely transcribed using an iScript™ cDNA Synthesis Kit  at 25°C for 5 minutes, 42°C for 30 minutes, followed by 85°C for 5 minutes. The cDNA was used as a template for PCR amplification by iQ™ Supermix (Bio-Rad Laboratories). The primer sequences, optimal annealing temperature, optimal cycles and product sizes are as shown in CCL2, CCL5 and CX3CL1 protein levels were assayed in aorta and cell homogenates as well as mouse plasma and culture media using commercially available ELISA kits .6) were harvested and lysed at 4°C in radioimmunoprecipitation assay lysis buffer. Aorta was homogenized at 4°C in radioimmunoprecipitation assay lysis buffer. The cell lysate or tissue homogenates were clarified by centrifugation at 14,000×g for 10 min at 4°C. Protein concentration in the soluble fraction was determined by the Bradford method. Protein samples (50–100 µg) were separated by a 12% SDS-polyacrylamide gel and then transferred onto polyvinylidene difluoride membranes. Membranes were then washed, blocked, and probed overnight at 4°C with rabbit polyclonal anti-CX3CR1 , IκBα , β-actin antibodies  and mouse monoclonal anti-CSE antibody  respectively, followed by secondary antibody for 2 h with a 1∶2000 dilution of HRP-conjugated, goat anti-rabbit IgG or goat anti-mouse IgG (Santa Cruz Biotechnology). Membranes were washed and then incubated in SuperSignal™ West Pico chemiluminescent substrate  before exposure to X-ray films (Pierce). The intensity of bands was quantified using LabWorks™ Image Analysis software (UVP).Cultured cells  or PAG  at the same time as the commencement of fat feeding and were sacrificed 8 weeks after fat feeding. Mononuclear cell (MNC) fraction was isolated from 1 ml of whole mouse blood using centrifugation  with Ficoll-Paque Plus  as described previously 6/tube) were labelled with rabbit anti-mouse CX3CR1 antibody for 1 h at room temperature, followed by incubation for a further 45 min with secondary PE-labelled antibodies (BD Biosciences) at room temperature. A third 30 min incubation was then carried out with a FITC conjugated CD11b antibody (BD Pharmingen). Appropriate anti-rabbit IgG control (Abcam) and Rat IgG control (BD Pharmingen) were also tested for CX3CR1 and CD11b antibodies, respectively. CX3CR1+CD11b+ cells were identified by flow cytometry  using FlowJo 5.7.2 software. For each sample, 100 000 cells were acquired.Male apoE6/ml) was added to the upper compartment. The cell migration plate assembly was incubated at 37°C with 5% CO2 for 2 h. Migratory cells on the bottom of insert membrane were dissociated from the membrane when incubated with Cell Detachment Buffer. The cells that migrated into the medium in the lower chamber or detached from the membrane by Cell Detachment Buffer were subsequently lysed and detected by CyQuant GR dye. This green fluorescent dye exhibits strong fluorescence enhancement when bound to cellular nucleic acids. The intensity of fluorescence, which correlated with the number of migrated cells, was measured with a fluorescence plate reader using a 485/520 nm filter set. Results were expressed as fluorescence intensity, RFU (relative fluorescence unit), by subtracting background fluorescence in cells migrating to the medium without chemoattractant, from specific fluorescence in cells migrating to CX3CL1.Chemotaxis was evaluated with QCM™ Chemotaxis 5 µm 96-well Cell Migration Assay . Serum free medium or chemoattractant solution (150 µL) was added to the lower chamber and 100 µL of cell suspension (2×10450 was read on a 96-well microplate reader (Tecan Systems Inc.).Nuclear extracts from RAW264.7 or peritoneal cells were prepared using a Nuclear Extraction Kit, as described by the manufacturer . Protein concentrations in nuclear extracts were determined using the Bradford assay (Bio-Rad Laboratories). PPAR-γ or NF-κB p65 DNA binding activity in nuclear extracts was assayed in triplicate with TransAM PPAR-γ Transcription Factor Assay Kits (Active Motif) according to manufacturer’s instructions. The OD3  in 1.2 M HCl. The absorbance of the resulting solution (670 nm) was measured 10 min thereafter by spectrophotometry (Tecan Systems Inc.). H2S was calculated against a calibration curve of sodium hydrosulfide . Results were shown as plasma H2S concentration in µM.Aliquots (120 µl) of plasma were mixed with distilled water (100 µl), trichloroacetic acid , zinc acetate , N, N-dimethyl-p-phenylenediamine sulfate  in 7.2 M HCl and FeCl2S was measured by a sulfur-sensitive electrode  2S was calculated against a calibration curve of sodium hydrosulfide . Results were shown as plasma H2S concentration in µM.Plasma H2S synthesiszing activity in aorta homogenates was measured essentially as described elsewhere 2S, followed by 10% v/v trichloroacetic acid (250 µl) to denature the protein and stop the reaction. Subsequently, N, N-dimethyl-p-phenylenediamine sulfate  in 7.2 M HCl was added, immediately followed by FeCl3  in 1.2 M HCl. The absorbance of the resulting solution at 670 nm was measured by spectrophotometry (Tecan Systems Inc). The H2S concentration was calculated against a calibration curve of NaHS. Results were then corrected for the DNA content of the tissue sample and expressed as nmoles H2S formed/mg DNA.HUltrasound biomicroscopy was performed as previously described Mice were anesthetized, followed by left ventricle perfusion with PBS and 10% neutralized formalin at a constant pressure of 100 mmHg. Brachiocephalic arteries were removed with a piece of the aortic arch and the stump of the right subclavian artery still attached to aid orientation during histological processing. Morphometric studies were performed in brachiocephalic artery. Brachiocephalic arteries were embedded in paraffin. Five sections (5 µm) taken 30 µm apart from the brachiocephalic artery of every mouse  were stained with hematoxylin and eosin and then used for histological analysis (n = 6 mice in each group). Estimation of plaque lipid content used a valid method as previously described 2O and PBST. Endogenous peroxidase activity was inactivated by treatment in 3% H2O2 for 15 min, followed by a wash in PBST. Nonspecific staining was blocked by incubation with Ultra-V block (Labvision) for 5 min. Slides were incubated with rabbit anti-mouse CX3CR1 antibody (Abcam) in 1∶100 dilution for 2 h at 37°C, followed by secondary antibody in a 1∶200 dilution of HRP-conjugated, goat anti-rabbit IgG (Santa Cruz Biotechnology) for 2 h at room temperature. Immunohistochemical staining was developed by exposure to 3, 3′-diaminobenzidine and counterstaining was performed with hematoxylin. The staining was examined by light microscopy using a light microscope . For fluorescent immunohistochemical staining, sections were incubated with rabbit anti-mouse CX3CR1 antibody  and rat anti mouse F4/80 antibody  overnight at 4°C, followed by incubation for 2 hour with fluorescently (Alexa Fluor 488 or Alexa Fluor 594) labeled secondary antibodies . Sections were then mounted and visualized using a fluorescent microscope (Olympus).Brachiocephalic arteries were deparaffinized in xylene and rehydrated in aqueous solutions with decreasing alcohol content, followed by a wash in PBST . Antigen retrieval was achieved by heating the slides in 10 mM sodium citrate (pH 6.0) at 95°C for 20 min. Slides were cooled for 20 min and washed in HThe data were expressed as mean ± SEM. The significance of differences among groups was evaluated by analysis of variance (ANOVA) with the post-hoc Tukey’s test when comparing three or more groups. The significance of differences between two groups was evaluated by the Student’s t-test. A P < 0.05 was regarded as statistically significant.Figure S1Effect of NaHS on CX3CR1 expression in mouse peritoneal macrophages stimulated with IFN-γ or LPS. RT-PCR analysis for CX3CR1 mRNA (A) and western blot analysis (B) for CX3CR1 protein expression were carried out as described in Materials and Methods. The data are means ± SEM of at least three independent experiments. *P<0.05, compared with unstimulated cells (control). ‡P<0.05, compared with stimulated cell pretreated with saline.(TIF)Click here for additional data file.Figure S2Effect of NaHS (100 µM) on chemotactic response to increasing doses of CX3CL1 in RAW264.7 cells stimulated with IFN-γ- or LPS. Chemotaxis of RAW264.7 cells towards CX3CL1 was assayed as described in Materials and Methods. The data are means ± SEM of at least three independent experiments. *P<0.05, compared with saline-pretreated, unstimulated cells (CX3CL1 10 ng/ml). **P<0.05, compared with saline-pretreated, stimulated cells (CX3CL1 10 ng/ml). †P<0.05, compared with saline-pretreated, unstimulated cells (CX3CL1 at the same concentration). ‡P<0.05, saline-pretreated, stimulated cells (CX3CL1 at the same concentration).(TIF)Click here for additional data file.Figure S3Effect of pretreatment with GW9662 on NaHS-induced downregulation of CX3CR1 expression and NaHS-induced inhibition of CX3CR1-mediated chemotaxis in IFN-γ or LPS-stimulated mouse peritoneal macrophages. Cells were incubated with GW9662 (10 µM) or vehicle for 1 hour, further incubated with NaHS (100 µM) or saline for 6 hours and then stimulated with IFN-γ (500 U/ml) or LPS (10 µg/ml) for 12 hours in the continuous presence of NaHS or saline. Western blot analysis for CX3CR1 expression (A) and chemotaxis towards CX3CL1 (50 ng/ml) (B) were assayed as described in Materials and Methods. The data are means ± SEM of at least three independent experiments. *P<0.05, compared with unstimulated cells (control). †P<0.05, compared with stimulated cells treated with saline and vehicle. ‡P<0.05, compared with stimulated cell treated with NaHS and vehicle.(TIF)Click here for additional data file.Figure S42S synthesizing activity (B) was assayed as described in Materials and Methods. The data are means ± SEM of four independent experiments. *P<0.05, compared with control cells. †P<0.05, compared with cells transfected with empty vector.Transfection of CSE cDNA construct in RAW264.7 cell. Cells were transfected with CSE cDNA construct or an identical empty vector lacking a cDNA insert as a control. The expression of CSE was verified by western blot analysis (A) and the H(TIF)Click here for additional data file.Figure S5Effect of NaHS or CSE overexpression on CX3CL1 mRNA expression and IκBα content in macrophages stimulated with IFN-γ or LPS. (A) Mouse peritoneal macrophages were pre-incubated with saline or NaHS (100 µM) for 6 hours and then stimulated with IFN-γ or LPS for 12 hours in the continuous presence of NaHS or saline. (B) RAW264.7 cells were transfected with CSE cDNA construct or empty vector and then were stimulated with IFN-γ or LPS for 12 hours. RT-PCR analysis for CX3CL1 mRNA and western blot analysis for IκBα content were carried out as described in Materials and Methods.(TIF)Click here for additional data file.Figure S6Effect of NaHS on CCR2 and CCR5 mRNA expression in macrophages stimulated with IFN-γ or LPS. RAW264.7 cells  or mouse peritoneal macrophages  were pre-incubated with saline or NaHS  for 6 hours and then stimulated with IFN-γ or LPS for 12 hours in the continuous presence of NaHS or saline. RT-PCR analysis for CCR2 and CCR5 mRNA was carried out as described in Materials and Methods.(TIF)Click here for additional data file.Figure S72S level during the development of atherosclerosis in fat-fed apoE−/− mice. Plasma H2S levels were assayed at indicated time points  by sulfur-sensitive electrode method. Results shown are the mean ± SEM . *P<0.05, compared with the basal level at 0 weeks. #P<0.05, compared with mice sacrificed 4 weeks after fat feeding. & P<0.05, compared with mice sacrificed 8 weeks after fat feeding.Alterations in plasma H(TIF)Click here for additional data file.Figure S8Negative control immunofluorescent staining in BCA with mouse IgG1 isotype control and Alexa Fluor 488 or Alexa Fluor 594 labeled secondary antibodies. Scale bar for histological images  =  20 µm.(TIF)Click here for additional data file.Figure S92S on CX3CR1 expression and CX3CL1-mediated chemotaxis of circulating monocytes isolated from fat-fed apoE−/− mice. ApoE−/− mice (n = 10 in each group) were fed a high-fat diet and at the same time received saline, NaHS , or PAG  and sacrificed 8 weeks after fat feeding. Whole blood was collected and the mononuclear cell fraction was isolated, and then incubated with antibodies to detect CD11b+CX3CR1+ cell populations using flow cytometry (A). CX3CR1 mean fluorescence intensity (B) and CX3CL1-induced chemotaxis (C) were determined among treatment groups. Results are expressed as mean±SEM. *P<0.05, compared with chow+saline group.Effect of H(TIF)Click here for additional data file.Table S1Effect of NaHS on CX3CL1 level in RAW264.7culture media stimulated with IFN-γ or LPS.(DOC)Click here for additional data file.Table S2Effect of NaHS on CX3CL1 in stimulated mouse peritoneal macrophages.(DOC)Click here for additional data file.Table S3Effect of CSE overexpression on CX3CL1 in stimulated RAW264.7 cells.(DOC)Click here for additional data file.Table S42S on CCL2 and CCL5 in stimulated RAW264.7.Effect of H(DOC)Click here for additional data file.Table S5Effect of treatment with NaHS on blood pressure and plasma lipids.(DOC)Click here for additional data file.Table S6Effect of treatment with PAG on blood pressure and plasma lipids.(DOC)Click here for additional data file.Table S72S on CCL2, CCL5, CCR2 and CCR5 in vivo.Effect of H(DOC)Click here for additional data file.Table S8Effect of PAG on CCL2, CCL5, CCR2 and CCR5 in vivo.(DOC)Click here for additional data file.


The mechanism of cell-cell contact dependent regulation of pericellular proteolysis in angiogenesis was examined by studying the expression of MMPs using isolated HUVECs in culture. Zymography, Immunoblot and RT-PCR analysis showed that the production and secretion of matrixmetalloproteinase-2 and matrixmetalloproteinase-9 by HUVECs in culture were high when they remain as individual cells and significantly decreased during later stages of culture when cells developed cell-cell contact and tubular network-like structure. As MMPs decreased there was significant upregulation of VE-cadherin in cells undergoing angiogenic transition. Investigations to understand the signaling pathways downstream of VE-cadherin showed a relatively high level of β-catenin in the nucleus of endothelial cells in culture during initial stages and decrease in its levels in the nucleus, associated with an increase in the cytosol during later stages of culture. The distribution of β-catenin was found to be regulated by Tyr/Ser phosphorylation status of this protein. Cell-cell contact dependent downregulation of MMPs during angiogenesis was also observed in experiments using proangiogenic substances which caused a rapid rate of downregulation of MMP-2 and MMP-9 and absence of downregulation of MMPs when treated with anti-angiogenic agents. The ability of ECs  and their supporting cellular elements to modify their immediate surrounding extracellular matrix (ECM) responding to multiple signals received from the environment is critically important during angiogenesis ,2. Both A temporal relation between the production of MMPs and the onset of angiogenesis has been reported . Down reApart from MMPs, cell adhesion molecules are also equally important regulators of angiogenesis. They form intercellular junctions between endothelial cells, which give the endothelium the ability to control the passage of solutes and circulating cells  and endoMCDB131-medium, gentamycin, antibiotic-antimycotic solution, gelatin, ortho-dianisidine, DEPC, curcumin, ursolic acid, gelatin, FCS, diamino benzidine, Tris, glycine, bovine serum albumin, protein A-sepharose beads, antibodies to MMP-2 (M4065), MMP-9 (M5427), VE-cadherin (C1821), β-catenin (C2206), CD 31 (C7714), CD 14 (C7673), CD 71 (C2063), phospho tyrosine (P5872), phospho serine (P5747), HRP  and FITC (F6397) conjugated secondary antibody were purchased from M/s Sigma Aldrich Co . NC membrane was from BioRad laboratories, Hercules, CA. ELISA and tissue culture plates were from NUNC A/S . Perfect RNA mini isolation kits and C-Master RT Plus PCR kits were purchased from Eppendorf .6 cells per well in MCDB 131 medium were seeded in NUNC multi-well plates and allowed to attach for 5 hrs, unattached cells were removed, fresh serum-free medium was added and maintained in culture overnight before starting the experiment. The purity of the cultures was assessed by checking for possible contamination with other cell population like monocyte/macrophage by monitoring expression of CD 14 and CD 71 by immunocytochemical technique. The cultures were negative for both the CD markers. Further, FACS analysis of the cultures showed that about 99.8% cells were CD 31 positive. All the experiments were performed according to the guidelines of the Institutional Ethical Committee.Endothelial cells were isolated by collagenase perfusion of umbilical vein as described before . The yieIndirect ELISA was performed using specific antibody . Cell cuTotal RNA was isolated from HUVECs using Perfect RNA Mini isolation kits procured from Eppendorf according to manufacturer's instruction. The primer pairs for human MMP-2, MMP-9, VE-cadherin, β-catenin and GAPDH were as follows; MMP-2 (195 bp) Forward 5'ATGGGGAATCGGTTGAAGG3' Reverse 5'AATTGCATTTCCTGACAGAAGG3'; MMP-9 (147 bp) Forward 5'GACTTGGCAGTGGAGACTGCGGGCA3' Reverse 5'GACCCCACCCC TCCTTGACAGGCAA3'; VE-cadherin (149 bp) Forward 5'GCACCAGTTTGGCCAATATA 3' Reverse 5'GGGTTTTTGCATAATAAGCAGG3'; β-catenin (291 bp); Forward 5'TTTTTAAA GGCAAGAATGCCTCA3' Reverse 5'CATTAATGAAGGCAAGTAGCCCA3' and GAPDH (680 bp) Forward 5'CGGAGTCAACGGATTTGGTCGTAT3' Reverse 5'GCAGGTCAGGTCC ACCACTAGC3'. The primer sequences were taken from NCBI nucleotide database and custom synthesized by Sigma Aldrich Chemicals Bangalore, India. RT-PCR was performed in an Eppendorf thermocycler, using the C-Master RT Plus PCR kit . 20 μl (2 μg) of the isolated RNA was used as template for reverse transcription and amplification as described before .Medium was removed, cells were washed with phosphate buffered saline (PBS), scraped from the culture plates, centrifuged and the cell pellets were lysed in Laemmli sample buffer. Protein equivalent amount of the lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels. Proteins were transferred to the nitrocellulose (NC) membrane using a semidry blotting apparatus, probed with specific antibody and developed using HRP-conjugated secondary antibody . Appropr2, pH 7.5) at 37°C for 24 hrs. The gels were stained with Coomassie brilliant blue R 250 and destained with water. Gelatinolytic activities appearing as clear zone were quantitated using Quantity One - 4.5.0 - Software (BioRad).The activity of MMPs secreted by HUVECs into the medium maintained in culture was determined by gelatin zymography . Zymogra2, 0.5 mM CaCl2, 0.1 mM PMSF, and protease inhibitors) [g for 10 min to pellet the nuclei. The cytosolic supernatant was centrifuged for 60 min at 13000 × g at 4°C. Protein equivalent amount of cytosolic and nuclear fractions were resolved on SDS polyacrylamide gels and immunoblotted to analyse the levels of β-catenin in nucleus and cytosolic fraction. For immunoprecipitation, equivalent amount of cellular proteins were brought to a volume of 1.0 ml in buffer containing 50 mM Tris-HCl, pH 7.5, 0.4 M NaCl, 5 mM EDTA and 0.5% NP-40. The clear supernatant was incubated with the anti-β-Catenin antibody for 2 hrs on ice followed by Protein A/G-Sepharose for immunoprecipitation. Beads were washed 3 times with the same buffer and extracted in Laemmli sample buffer; subjected to SDS-PAGE, electroblotted and probed with monoclonal antibodies against phosphotyrosine and phosphoserine.Cell pellets were resuspended in 500 μl PBS ice-cold nuclear buffer  . The susThe statistical significance of difference was analysed by Duncan's one way Analysis of Variance (ANOVA) using SPSS10 Software.th day was less than 40% of that on the 1st day. Although there was no significant difference in the activity of MMP-2 and MMP-9 on the 2nd day when compared to that on the 1st day, the levels of the proteins were about 10-15% more on the 2nd day. RT-PCR analysis was done to confirm whether the decrease in the activity of MMP-2  is reported to inhibit capillary network-like structure formation and angiogenesis . The relIn order to understand whether there is any relation between the cell-cell contact formation and MMP production, the kinetics of production of VE-cadherin, the intercellular cell adhesion molecule involved in endothelial cell-cell interaction and angiogenesis was analysed by western blotting. There was significant increase in the amount of this protein with the progression of culture which on the fourth day was about 4 fold more than that on the first day Figure . The expβ-catenin is one of the accessory proteins associated with the cytoplasmic tail of VE-cadherin aiding the stabilization of cell-cell adhesion. When unbound to VE-cadherin, β-catenin from cytoplasm can translocate into the nucleus. The relative levels of this protein in the cytosol and nucleus were analysed by western blotting and the results are shown in Figure Inorder to further examine the relation between the production of cell-adhesion molecules and MMPs by ECs during angiogenesis, changes in cadherin and catenin with the progression of culture were studied in cells treated with curcumin under serum free conditions. The results are given in Figure Analysis of the relative levels of β-catenin in the nucleus and cytoplasm of the cells treated with curcumin showed relatively higher levels of this protein in the nucleus during initial stages (upto 48 hrs) which declined thereafter concomitantly with significant increase in its levels in the cytosol during the later stages of culture Figure . In cellIn order to understand the phosphorylation status of β-catenin, the trigger that directs the nuclear translocation of cytosolic β-catenin, it was immunoprecipitated and probed with specific phospho-antibody and the results are shown in Figure rd day in culture was significantly less than that on the first day and that of serine phosphorylation was more on third day than the first day in culture in serum free condition. Since there was difference in the extent of phosphorylation among different treatments, the intensity of each was expressed as percentage of the sum of the intensities of bands corresponding to P-Tyr and P-Ser. The relative levels of tyrosine phosphorylation to serine phosphorylation of β-catenin was high during the initial stages of culture which decreased as the level of serine phosphorylation increased during later stages of culture in control and curcumin treated cells. But the ratio of tyrosine phosphorylation to serine phosphorylation of β-catenin was observed to be 1:1 in cells treated with curcumin in presence of serum which remained unaffected throughout the culture.The changes in the phosphorylation of tyrosine and serine in β-catenin in cells treated with curcumin in serum free and serum supplemented conditions for different periods were also studied. The extent of tyrosine phosphorylation of β-catenin on 3To examine whether the activation of β-catenin is associated with the changes in MMPs, the effect of inhibition of the glycogen synthase kinase-3 (GSK-3), an enzyme that catalyses serine phosphorylation of β-catenin by lithium on the activity of MMPs was analysed. In cells treated with Li, no downregulation of MMPs was observed and there was significant increase in the activity of both MMP-2 and MMP-9 when compared to the respective control  by ECs . OpposinThe downregulation of MMPs minimizes peri-cellular proteolysis and facilitates the cell-cell/cell-matrix interactions which are vital for the maturation and stabilization of neo-vessels. It appears to be endothelial cell-specific and is related to the angiogenic transition associated with the formation of cell-cell contact. Other cell systems such as monoctye/macrophage , hepaticDuring the initial stages when cells remained mostly as individual ones, there was more MMP-2 and MMP-9 and less VE-cadherin and at later stages when grouping of cells and network-like structures developed, increase in VE-cadherin and decrease in MMP-2 and MMP-9 production were observed. Further, curcumin that accelerated angiogenic phenotype in HUVECs under serum free conditions caused a significant upregulation of VE-cadherin and downregulation of MMPs. Although direct proof relating cell-cell contact formation and increase in the levels and distribution of VE-cadherin is not provided in our present study, there are reports showing change in cell-cell contact with change in VE-cadherin expression ,42. It hFurther investigations to understand the signaling pathways downstream of VE-cadherin suggested the involvement of β-catenin. There was a relatively high level of β-catenin in the nucleus of ECs in culture during initial stages and decrease in its levels in the nucleus associated with an increase in the cytosol during later stages. Apart from contributing to stabilise cell-cell contact through cadherin-catenin association, the translocation of catenin can result in changes in the expression of catenin responsive genes. The activity of β-catenin is regulated by the phosphorylation status of this protein. Our results on increased tyrosine phosphorylation and relatively higher levels of β-catenin in the nucleus during initial stages (upto 48 hrs) and relative increase in serine phosphorylation and decreased nuclear distribution at later stages are consistent with the reported role of phosphorylation of β-catenin in its translocation from cytosol to nucleus ,45. FurtThere was correlation between the distribution of β-catenin in the nucleus and the expression of MMP-2 and MMP-9. As β-catenin in the nucleus decreased with the progression of the culture, the expression of MMPs also decreased. Such a down regulation of MMPs was not observed on treatment with Li which caused increase in the nuclear distribution of β-catenin. The down regulation of MMP-2 and MMP-9 caused by curcumin was also reversed by Li. The effect of Li on the distribution of β-catenin may be related to its phoshorylation status as Li is known to inhibit GSK-3 which catalyses phosphorylation of serine in catenin. Similar results were also obtained on treatment of cells with SB216763 a specific inhibitor of GSK-3 (data not shown). However, the possibility of these substances acting on other intracellular pathways cannot be excluded. Although these results show a correlation between phosphorylation status and nuclear distribution of β-catenin and the levels of MMPs it is not clear whether the expression of MMP-2 and MMP-9 in HUVECs is dependent on VE cadherin-β-catenin signaling. MMP genes are under the influence of the transcriptional activity of β-catenin as β-catenin responsive elements are reported to be present in the MMP genes ,47. AlthAlthough reciprocal changes in the expression of MMPs and VE-cadherin were observed in HUVECs in these different experiments where both pro-angiogenic and anti-angiogenic substances were tested, it is possible that these changes may as well be unrelated.As MMP level decrease probably by other unknown mechanisms pericellular proteolysis is reduced resulting in the retention of VE-cadherin and other cell adhesion molecules promoting cell-cell contact formation. More direct experiments would be required to confirm this relationship between the expression of the cell adhesion molecule in angiogeneiss and modulation of MMP expression.The authors declare that they have no competing interests.Most of the experiments were performed by MSK. All the authors contributed to analysis and interpretation of data, discussion and preparation of the manuscript. All the authors read and approved the final manuscript.Immunocytochemical analysis of the expression and translocation of β-catenin in HUVECs maintained in culture for 24 hrs. Figure S1 Expression and translocation of β-catenin in HUVECs: Day1 HUVECs were cultured in MCDB 131 medium and maintained in culture for 5 days. Cells were harvested at Day 1 (24 hrs) immunocytochemical analysis was performed to analyze the expression of β-catenin. Left panel Top to bottom (a) microphotograph of cells Day 1 (40 X) stained with β-catenin, (b) overlaid microphotograph of cells with nucleus stained (Hoechst stain), (c) phase contrast image of cells Day 1. Right panel top to bottom, (a) microphotograph of cells Day 1 (100 X) stained with β-catenin, (b) overlaid image with nucleus stained (Hoechst stain) and (c) microphograph of cells with nucleus stained. Arrow heads to the β-catenin stained red, nucleus stained blue. The β-catenin is located in nucleus of the cells during day 1 where the cells are in single spherical morphology and no cell-cell contact formation observed.Click here for fileImmunocytochemical analysis of the expression and translocation of β-catenin in HUVECs maintained in culture for 120 hrs. Figure S2 Expression and translocation of β-catenin in HUVECs: Day 5HUVECs were cultured in MCDB 131 medium and maintained in culture for 5 days (120 hrs). Cells were harvested at Day 5, and immunocytochemical analysis was performed to analyze the expression of β-catenin. Left panel Top to bottom microphotograph of cells Day 5 (40 X) stained with β-catenin, (b) overlaid image of cells with nucleus stained (Hoechst stain) (c) phase contrast image and (d) microphotograph of cells with nucleus stained (Hoechst stain). Right panel Top to bottom, (a) microphotograph of cells Day 5 (100 X) stained with β-catenin, (b) microphotograph of cells with nucleus stained and (c) overlaid microphotograph with nucleus stained (Hoechst stain). Arrow heads to the β-catenin stained red, nucleus stained blue. HUVECs formed extensive cell-cell contact and capillary network like structure. The β-catenin is located at the cell contact sites.Click here for file


Aberrant Wnt signalling is implicated in numerous human cancers, and understanding the effects of modulation of pathway members may lead to the development of novel therapeutics. Expression of secreted frizzled related protein 4 (SFRP4), an extracellular modulator of the Wnt signalling pathway, is progressively lost in more aggressive ovarian cancer phenotypes. Here we show that recombinant SFRP4 (rSFRP4) treatment of a serous ovarian cancer cell line results in inhibition of β-catenin dependent Wnt signalling as measured by TOP/FOP Wnt reporter assay and decreased transcription of Wnt target genes, Axin2, CyclinD1 and Myc. In addition, rSFRP4 treatment significantly increased the ability of ovarian cancer cells to adhere to collagen and fibronectin, and decreased their ability to migrate across an inflicted wound. We conclude that these changes in cell behaviour may be mediated via mesenchymal to epithelial transition (MET), as rSFRP4 treatment also resulted in increased expression of the epithelial marker E-cadherin, and reduced expression of Vimentin and Twist. Combined, these results indicate that modulation of a single upstream gatekeeper of Wnt signalling can have effects on downstream Wnt signalling and ovarian cancer cell behaviour, as mediated through epithelial to mesenchymal plasticity (EMP). This raises the possibility that SFRP4 may be used both diagnostically and therapeutically in epithelial ovarian cancer. Epithelial ovarian cancer has the highest mortality of all female gynaecological cancers The Wnt signalling pathway is a crucial developmental pathway involved in differentiation, polarity, migration, invasion, adhesion and survival MYC), CyclinD1 (CCND1) and Axin2 (AXIN2) This multifaceted signalling network is traditionally simplified by dividing the network into canonical (β-catenin dependent) and non-canonical (β-catenin independent) pathways. Canonical Wnt signalling involves the binding of Wnt ligand to one of ten Frizzled receptors (Fzd) in the presence of a low density lipoprotein receptor related protein (LRP) co-receptor. This generates a cascade of events leading to the disassembly of the Axin/APC/GSK3β destruction complex and the stabilisation of β-catenin. Accumulation of β-catenin in the cytoplasm results in translocation to the nucleus and TCF/LEF mediated activation of target genes involved in cell differentiation and proliferation. Key downstream targets of activated canonical Wnt signalling include C-myc , WIF-1 and the family of secreted frizzled receptor proteins (SFRP1-5). SFRPs are soluble extracellular proteins characterized by their frizzled-like cysteine rich domain (CRD), which is thought to be responsible for their ability to modulate Wnt signalling by binding directly to Wnt ligands or Frizzled receptors. SFRPs have been shown to function as tumour suppressors in other cancer types Epithelial to mesenchymal transition (EMT) is a key developmental process that cancer cells hijack to increase their aggressiveness and invasive potential In this present study we investigate the functional effects of modulation of a key Wnt pathway gatekeeper, SFRP4 on Wnt signalling, cell behaviour and EMT. We report for the first time that re-expression of SFRP4 in an epithelial ovarian cancer cell line inhibits Wnt signalling, increases adhesion, inhibits cell migration and inhibits EMT. Since SFRP4 expression has been shown to be lost in a large majority of ovarian cancer patients 2, at 37°C and were demonstrated to be free of mycoplasma contamination.Human serous ovarian cancer OVCAR3 cells, obtained from ATCC  were cultured in RPMI containing 10% fetal calf serum. Media was supplemented with penicillin/streptomycin  and 1.8 mM GlutaMAX (Life Technologies). All cells were grown in a humidified atmosphere of 5% COOVCAR3 cells were plated at a concentration of 5000 cells/well on white bottomed 96 well plates. Cells were serum starved overnight and co-transfected with 0.2 µg of either TOPflash (3× TCF4 binding sites) or FOPflash (3× mutated TCF4 binding sites) expression plasmids , and 0.1 µg pRL-TK  as a control, using Lipofectamine2000. Cells were subsequently treated with increasing doses of rSFRP4 and/or recombinant Wnt3 a (rWnt3 a 0.1 µg/ml) for 48 hours prior to luciferase activities being measured using a Glomax 96 Microplate Luminometer . Firefly luciferase activity was normalized for transfection efficiency by dividing by the Renilla luciferase activity. The TOP/FOP ratio was used as a measure of β-catenin driven transcription. Average activity and standard deviations were derived from octopulate transfected samples.Following DNAse treatment, 1 µg of total RNA was transcribed to cDNA using the Quantitect RT kit . Controls containing RNA but without reverse transcriptase were included for all samples. qPCR was performed in triplicate on a Stratagene MxPro 3005 P machine using 25 ng of cDNA template and SYBR green/Rox master mix (Qiagen). Expression was normalised to three different housekeeping genes  using the Vandesompele normalisation method TGTGTTACGAGTGGCG 3′, reverse (R) 5′ GGGGGATTACTACGACTG 3′, CDH1: F 5′ AGGCCAAGCAGCAGTACATT 3′ R 5′ ATTCACATCCAGCACATCCA 3′, VIM F 5′ CCAAACTTTTCCTCCCTGAACC 3′ R 5′ GTGATGCTGAGAAGTTTCGTTGA 3′, TWIST F 5′ GCCAATCAGCCACTGAAAGG 3′ R 5′ TGTTCTTATAGTTCCTCTGATTGTTACCA 3′, AXIN2 F 5′ TCAAGTGCAAACTTTCGCCAACC 3′ R 5′ TAGCCAGAACCTATGTGATAAGG 3′, MYC F 5′ CGTCTCCACACATCAGCACAA 3′, R 5′ CACTGTCCAACTTGACCCTCTTG 3′, CCND1 F 5′ GGCGGAGGAGAACAAACAGA 3′ R 5′ TGGCACAAGAGGCAACGA 3′, JNK F 5′TCTGGTATGATCCTTCTGAAGCA 3′ R 5′ TCCTCCAAGTCCATAACTTCCTT, RHOA F 5′ GGAAAGCAGGTAGAGTTGGCT 3′ R 5′ GGCTGTCGATGGAAAAACACAT 3′, RAC1 F 5′ TGTAGTCGCTTTGCCTATTGATG 3′ R 5′ CATCGTCAGCACTAGCACAGTTT 3′, PRKCA F 5′ GCTTCCAGTGCCAAGTTTGC 3′ R 5′ CATCGTCAGCACTAGCACAGTTT 3′, SDHA F 5′ CTTGAATGAGGCTGACTGTG 3′ R 5′ ATCACATAAGCTGGTCCTGT 3′, HSPCB F 5′ TCTGGGTATCGGAAAGCAAGCC 3′ R 5′ GTGCACTTCCTCAGGCATCTTG 3′, YWHZA F 5′ ACTTTTGGTACATTGTGGCTTCAA 3′ R 5′ CCGCCAGGACAAACCAGTAT 3′.Primer sequences used for qPCR were SFRP4: forward (F) 5′ Cell lysates were prepared by lysing cells in lysis buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 0.27 M sucrose, 1× complete protease inhibitor ). Lysates were centrifuged and the supernatant was collected for protein concentration analysis using the BCA kit . Nuclear protein lysates were prepared by lysing cells in ice-cold Nuclei Buffer  and incubating on ice for 10 minutes. Nuclei were recovered by centrifugation at 900g for 3 min, washed in Nuclei Wash Buffer  and resuspended in lysis buffer. Proteins were separated using SDS–PAGE and transferred to PVDF membranes. For Western blotting, antibodies against SFRP4 , E-cadherin , Vimentin , Twist , β-catenin , Histone H3  and α-Tubulin  were used. Protein bands were detected and quantified using the Image Quant LAS 4000 .OVCAR3 cells were seeded onto IBIDI Culture-Inserts  and were treated for 24 hours with rSFRP4 (5 µg/ml). IBIDI inserts were removed and closure of the resulting wound was monitored over the next 48 hours. Images of the wound were captured at different time points using the Leica DMIL microscope system .Tissue culture plates were coated with solutions of 10 µg/ml type I collagen , 5 µg/ml fibronectin  or 3% BSA in PBS and were incubated overnight at 37°C. Plates were rinsed with 80% ethanol, incubated in 3% BSA in serum free media for 30 min at 37°C and rinsed again with PBS. Cell suspensions were added to the coated plates and allowed to adhere for 4 hours at 37°C. Plates were gently washed with PBS, fixed with 96% ethanol and stained with 0.1% crystal violet. Excess stain was removed by extensively washing plates with sterile water. After plates had dried, cells were lysed with 50% acetic acid and absorbance was read at 595 nm using a SpectraMax Plus384 Absorbance Microplate Reader .P<0.05, **P<0.01, ***P<0.001.Results are expressed as mean ± standard deviation (SD). Statistics were performed using a two-tailed student's t test. *in vitro could inhibit β-catenin dependent Wnt signalling. We selected the serous ovarian cancer cell line OVCAR3 for these experiments as it lacks any detectable SFRP4 expression and was previously reported to exhibit constitutive Wnt signalling . In contrast to CCND1 and MYC, AXIN2 is well accepted as a specific Wnt target gene, and to date has not been linked to other signalling pathways Forty eight hours of treatment with rSFRP4 (5 µg/ml) lead to an approximate two fold increase in SFRP4 mRNA and protein, as the addition of extracellular recombinant Wnt proteins enhances endogenous protein production. . The expCombined, these initial experiments demonstrate that the addition of a single Wnt pathway modulator can modulate gene transcription in a model serous ovarian cancer cell line. Based on the TOP/FOP Wnt reporter assay, Western Blots and Wnt target gene results, our data also suggest that in the case of ovarian cancer, SFRP4 does indeed function as an antagonist of the canonical or β-catenin dependent Wnt signalling pathway. It also appears that SFRP4 does not act to inhibit β-catenin independent Wnt signalling, as four target genes were unaffected by treatment with SFRP4. This is of interest, as it is unclear in the field of Wnt signalling exactly which Wnt ligands are inhibited by which specific members of the SFRP family, and whether in some cases SFRPs may actually augment rather than inhibit Wnt signalling in vitro, which fits with our previous clinical data reporting that patients expressing SFRP4 had better progression free and overall survival compared with those lacking SFRP4 expression SFRP4 treatment had no effect on cellular proliferation , but sigAs changes in cellular behaviour are linked to EMT, we also investigated whether SFRP4 would affect cell morphology and the expression of the key EMT markers, E-cadherin (CDH1), Vimentin (VIM), and Twist1 (TWIST1). While no obvious changes in cell morphology were noted 48 hours after SFRP4 treatment , treatmeEMT, like the Wnt pathway, is most strongly linked to development, yet recently has received widespread interest in its potential as a target for cancer therapy. Generalised processes that occur in all cancer cells are an attractive avenue for cancer therapy, as potential treatments will have widespread applicability. Additionally, aberrant Wnt signalling has been strongly linked to cancers with a poor prognosis The Wnt signalling pathway is a highly complex and interconnected pathway linked to many human diseases. As the pathway is mainly involved in embryogenesis and adult tissue repair, drugs targeted to this pathway are not expected to cause significant side effects or toxicity


SGO1 was expressed from germinal vesicle (GV) to the metaphase II stage. SGO1 accumulated on condensed and scattered chromosomes from pre-metaphase I to metaphase II. The over-expression of SGO1 did not interfere with the process of homologous chromosome separation, although once separated they were unable to move to the opposing spindle poles. This often resulted in the formation of oocytes with 60 replicated chromosomes. Depletion of SGO1 in GV oocytes affected chromosomal separation resulting in abnormal chromosome alignment at a significantly higher proportion than in control oocytes. Knockdown of SGO1 expression significantly decreased the embryonic developmental rate and quality. To further confirm the function(s) of SGO1 during mitosis, bovine embryonic fibroblast cells were transfected with SGO1 siRNAs. SGO1 depletion induced the premature dissociation of chromosomal cohesion at the centromere and along the chromosome arm giving rise to abnormal appearing mitotic patterns. The results of this study infer that SGO1 is involved in the centromeric cohesion of sister chromatids and chromosomal movement towards the spindle poles. Depletion of SGO1 causes arrestment of cell division in meiosis and mitosis.Shugoshin (SGO) is a critical factor that enforces cohesion from segregation of paired sister chromatids during mitosis and meiosis. It has been studied mainly in invertebrates. Knowledge of SGO(s) in a mammalian system has only been reported in the mouse and Hela cells. In this study, the functions of SGO1 in bovine oocytes during meiotic maturation, early embryonic development and somatic cell mitosis were investigated. The results showed that  Meiosis is a unique genetic phenomenon in which DNA replicates once followed by two sequential cell divisions. During the first meiotic division (meiosis I), the homologous chromosomes are paired and crossing over occurs with the formation of a chiasmata. Each pair of sister chromatids remain tightly linked until all chromosomes are aligned at the equatorial plate and attached to the meiotic spindle at metaphase via their kinetochores Xenopus laevis SGO1 (hSGO1) results in the removal of cohesins even as centromeres pass through the prophase pathway in vitro system, purified hSGO1 dephosphorylates the SA2 subunit of the cohesin complex from the originally phosphorylated state by the Polo-like Kinase 1(PLK1). Okadaic acid treatment will diminish the reaction SGO2 causes a mild defect in the centromeric protection of cohesin, and results in chromosomal mal-alignment defects Sgo2-knockout mice develop normally and embryonic fibroblast cells proliferate with no obvious mitotic defects Two members of the shugoshin protein family have been reported. Shugoshin 1 (SGO1) exists in fission yeast Sgo2-depleted mouse oocytes have shown that precociously separated chromatids were frequently observed at metaphase II, but not during meiosis I. These observations indicate that in oocytes, Sgo2 depletion abolishes sister centromatid cohesion during anaphase I Mouse SGO1 and SGO2 are ubiquitously expressed in proliferating cells, and SGO2 expression level has been reported to be higher in testis and oocytes SGO1 caused enhanced chromosomal instability and colon tumorigenesis in the mouse hSGO1 is lower in tumor tissues of colorectal cancer patients SGO1 RNAi has been reported to induce transformed cells into apoptosis Defects in the regulation of chromosome separation will result in arrestment of cell division, aneuploidy and tumorigenesis Much of the information obtained about mammalian SGO in meiosis has been obtained from mice. In this study we use the bovine model and investigate the roles of SGO1 during oocyte meiotic maturation and early embryonic development with exogenous over expression and by RNA interference. The role of bovine SGO1 in fibroblast cells during mitosis was also investigated and compared to the results obtained from the meiotic studies.All studies adhered to procedures consistent with the National Research Council Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Inner Mongolia University.Chemicals and medium used in this study were purchased from Sigma Chemical Co.  unless otherwise indicated. Primers were synthesized by Takara Biotechnology Dalian Co. Ltd . Sequencing assays were performed by Life Technologies Corporation.Bos taurus L.) oocytes was performed as previously described Maturation of bovine  were activated by 5 µM ionomycine for 5 min, then treated with 10 µg/mL cycloheximide (CHX) and 5 µg/mL CB in CR1aa (Charles Rosenkrans 1 amino acid) plus 3% FBS for 5 h at 38.5°C in 5% CO2 in air. Oocytes were then cultured in 40 µL droplets of CR1aa contained 0.3% bovine serum albumin (BSA) for 40 h. Cleaved embryos were transferred to CR1aa supplemented with 4% FBS medium and placed upon a feeder layer of bovine cumulus cells and incubated in 5% COSGO1 cDNA. Recognition sequences of restriction enzymes BamH I and Not I (underlined) were added up stream of the forward and reverse primers, respectively.Total RNA was extracted from 100 GV oocytes using a Fast RNA Micro Kit (Watson) and the first strand cDNA was generated with RT-PCR kit (Takara) using oligo (dT) primers. The following primers were designed according to the sequences in Genbank  and were used to clone the full length of GGATCCTGGTGTGAGCAGAGCAGCAG 3′F: 5′ GCGGCCGCTGTACTTGCTGCACATTTTT 3′R: 5′ SGO1 cDNA was inserted into the multiple clone site (MCS) of pCDNA3.1 (+) myc-hisB vector (Invitrogen) driven by a T7 promoter via Not I and BamH I. The downstream myc tag was used to detect SGO1 signals.The in vitro transcription reactions, the pCDNA3.1 (+)-SGO1- myc-hisB plasmid was linearized by restriction enzyme Dra III and purified by SV Gel and PCR Clean-up (promega). A T7 message machine kit (Ambion) was used to produce capped mRNA, which was then purified using the RNA clean kit (TIANGEN). The pCDNA3.1 (+) - myc-hisB plasmid was also linearized and performed to produce MYC mRNA as controls. The concentration of SGO1-MYC/MYC mRNA was detected by NANODROP 2000c , and then the mRNA was diluted into a lower concentration (0.3 mg/ml) for localization tracking and to a higher concentration (3.0 mg/ml) for overexpressing the protein. The forward and reverse primers incorporated into the SGO1 sequences and the plasmid sequences downstream of MYC tag are listed below. They were specifically designed to identify SGO1-MYC mRNA upon using RT-PCR.For the F: 5′ CAGTCCTAGAGCAGAAGATGGC3′,R: 5′GTGATGGTGATGATGACCGGTA3′.SGO1 mRNA interference sequences were designed via software published by Applied Biosystems official website (http://www.ambion.com/techlib/siRNA_finder.html). As shown in SGO1 siRNA  sequences and one pair of negative control siRNA sequences were synthesized . The siRNAs used in this study were a mixture of three pairs of SGO1 siRNA (m-siRNA). Small interfering RNAs were mixed equally before microinjecting into oocytes.Based upon information from GenBank, the SGO1-MYC mRNA solutions at concentrations of 0.3 µg/µl or 3.0 µg/µl were injected into the cytoplasm of GV oocytes. The same amount of MYC was injected as a control. The injected oocytes were arrested at the GV stage in 25 µM Roscovitine for 3 hours before meiotic resumption. 5 pl of SGO1 m-siRNAs solution were microinjected into the cytoplasm of GV intact oocytes and the injected oocytes were maintained in maturation medium containing 25 µM Roscovitine for 24 h before meiotic maturation. 5 pl of SGO1 m-siRNAs were also microinjected into the cytoplasm of parthenogenetic pseudo-zygotes which were held in CR1aa. The same amount of non-sense siRNA was injected for the negative control.Microinjection of oocytes was performed using an Eppendorf micro injector  and completed within 30 minutes per treatment group. The injector pipette was replaced after fifty oocytes were injected to avoid possible false negative results. For localization tracking or overexpression, SGO1 with the reference gene being GAPDH . SGO1, forward, CCAGTAGTGACGACAACTCCAGAGA; reverse, CTGGACAGTCAGCACCCTCAAG.Analysis of relative gene expression was measured by real-time quantitative PCR using the 2(-Delta Delta C (T)) method GAPDH, forward, GATGGTGAAGGTCGGAGTGAAC; reverse, GTCATTGATGGCGACGATGT. SYBR Premix Ex Taq II kit (Takara) was used for real-time PCR in an ABI prism 7300 Sequence Detection System . The steps involved 95°C 30 s, 40 cycles of 95°C 5 s and 60°C 31 s.Bos primigenius taurus) fibroblasts were obtained from the skin of a bovine fetus at day 50, and fibroblasts from passage 3 to passage 5 were used to perform transfections. Embryonic fibroblasts were cultured in DMEM (Dulbeccòs Modified Eagle Medium) supplemented with 10% FBS and 0.2 mM L-glutamine. Cell synchronization was performed as described Bovine on glass slides for 24 h to obtain chromosome spreads for analysis. Chromosome spreads were stained with 1% Giemsa for 10 min. Cells in mitosis were harvested by trypsinization and treated with 0.075 M KCl for 40 minutes at room temperature, then fixed by fresh methanol: glacial acetic acid (3∶1) for 30 minutes with three repeats. Fixed cell suspensions were dropped onto a glass slide and the slides were dried at room temperature for 24 h. Chromosomes were stained with 1% Giemsa for 20 min and the slides were washed slightly by tap water and dried for microscopic examination. Images were digitally captured using the Nikon Elements Program with a Nikon microscope .Data (mean ± SEM) were pooled from at least three replicates per experiment and analyzed by one-way analysis of variance (ANOVA) using origin 8 software. Data were analyzed using the Student t-test with a P-value of <0.05 being considered statistically significant.SGO1 during bovine oocyte meiotic maturation. Real-time RT PCR was performed to detect the mRNA level of SGO1 at 0, 8, 12, 16 and 24 h. The 2 (-Delta Delta C (T)) method was used for data analysis.This experiment was designed to examine the expression levels of SGO1-MYC mRNA that had been diluted to a lower concentration. Oocytes were collected at 0, 8, 14, 18 and 24 h of culture, which corresponds to GV, germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase/telophase I (AI/TI), and metaphase II (MII) stages. Subcellular distributions of SGO1-MYC were observed with a fluorescence microscope.This experiment was designed to examine the distribution of SGO1 during oocyte meiotic maturation. COCs were denuded and processed for injection of SGO1 during meiosis I affects chromosome separation. Oocytes at the GV stage were injected with SGO1-MYC mRNA at a higher concentration. After meiotic maturation, the oocytes were immuno-stained and analyzed. Concomitantly with the treatment of oocytes, chromosome spreading was performed and analyzed.This experiment determined whether over-expression of SGO1 at meiosis I affects chromosome separation. Oocytes at the GV stage were injected with SGO1-specific siRNA, followed by treatment of 25 µmol Roscovitine for 24 h to prevent meiotic resumption. After in vitro maturation for 24 h, chromosome spreading was performed and the percentage of chromosome anomalies was determined.This experiment determined whether RNAi of SGO1 in 1-cell embryos affects subsequent development. Zygotes obtained from parthenogenic activation were injected with SGO1-specific siRNA, followed by the observation of embryonic development in vitro. The cleavage and blastocyst rates were calculated. The cell number of individual blastocysts was determined.This experiment determined whether depletion of SGO1 affects chromosome separation during mitosis in bovine embryonic fibroblasts. Synchronized transfected cells were harvested at different times. Observations were classified according to particular chromosomal configuration patterns.This experiment determined whether RNAi of SGO1, oocytes were incubated in vitro for 0, 8, 12, 16, and 24 h, which corresponded to GV, germinal vesicle breakdown (GVBD), metaphase I (MI), anaphase I/telophase (AI/TI), and metaphase II (MII) stages, respectively SGO1 expression levels were detected by Real-time RT-PCR. SGO1 mRNA level at GV was normalized to 100%. SGO1 expression level decreased to 57.0% at GVBD, which was significantly lower than what was observed at GV. The amount of SGO1 mRNA then increased to 95% at MI and 132.0% at AI/TI. The mRNA amount in MII oocytes was 3.3-fold (331.7%) higher than in GV oocytes  in the treatment group as compared to the control (81.0%). The majority of treated oocytes did not extrude a PB1 (the first polar body). Upon further analysis of these oocytes without a PB1, the chromosomes aligned at the equatorial plate with irregular configurations  when compared to the non-sense injected (16.2%) and the non-injected groups (19.2%) (SGO1 RNAi group (average 65.0 cells) when compared to non-sense group (90.0 cells) and the non-injected groups (105.0 cells)  . Cell nu0 cells) . SGO1-spSGO1 siRNAs as shown in SGO1 was depleted, chromosomal alignment and chromosome spread appearance were classified into seven different pattern types . These anomalies were significantly higher than that in the non-sense depleted cells. It appears that SGO1 depletion causes a premature dissociation of chromosomal arm cohesions. Cells in anaphase at 9 h in non-sense treated cells emerged at 5 h in SGO1-depleted cells and increased over time , although only a small part of mitotic cells will normally entry anaphase precociously. SGO1 depletion induces the premature dissociation of cohesion along the chromosomal arms and at the centromere. Mitotic patterns 5 - 7 were only observed in SGO1- depleted cells and their associated chromosome anomalies increased significantly  reported that over expression of SGO1 in the budding yeast causes mitotic arrestment SGO1 in the bovine oocyte resulted in meiotic arrestment and a decrease in maturation rate. Therefore, it appears that bovine SGO1 is involved in the movement of chromosomes towards the poles. Excessive amounts of SGO1 somehow antagonize the pull of the microtubules preventing the formation of haploid gametes. SGO1 may simply be involved in tension sensing and setting at the centromere during mitosis as has been reported in Hela cells Xenopus extracts SGO1-depleted oocytes. SGO1 depletion causes oocytes to arrest during meiosis as the chromosomes fail to migrate to opposite spindle poles, thus thwarting normal separation and inducing premature chromatid separation. The observations also suggest that, as in other models It is generally accepted that during oocyte maturation, there is no transcription activity. Our quantative analysis results showed that bovine SGO1 depletion and classified into distinctive groups (shown in SGO1-depleted cells. Depletion of SGO1 caused dissociation of cohesion at the centromere on some or all chromosomal arms. This resulted in mitotic arrestment. Paired sister chromatids were pulled further apart from each other to the point they eventually formed hyper condensed and individually recognized chromosomes. This was probably the primary cause leading to the formation of polyploid cells. The effects on mitosis seemed to be similar to what we observed during meiosis. SGO1 protects centromeric cohesion, which in consistent with similar reports in Xenopus cells The mitotic processes in bovine fibroblast cells were analyzed in similar detail to the meiotic processes that occurred in the bovine oocyte/embryo. Seven unique mitotic configuration patterns were observed during shown in . PatternSGO1 will result in the arrestment of meiotic and mitotic cell division. SGO1 was essential for the faithful chromosomal separation.In conclusion, bovine SGO1 is associated with chromosomal polarization during meiosis and involves centromeric cohesion of sister chromatids during both meiosis and mitosis. Depletion of


In gliomagenesis, the role of canonical  signalling is largely unknown. Here, we studied canonical wnt pathway activation in 15 short-term cultures from high-grade gliomas and potential pathomechanisms leading to cytoplasmic β-catenin accumulation. Furthermore, we assessed the prognostic relevance of β-catenin expression in a tissue microarray comprising 283 astrocytomas. Expression of β-catenin, its transcriptional cofactors TCF-1 and TCF-4 as well as GSK-3β and APC, constituents of the β-catenin degradation complex was confirmed by RT-PCR in all cultures. A cytoplasmic β-catenin pool was detectable in 13/15 cultures leading to some transcriptional activity assessed by luciferase reporter gene assay in 8/13. Unlike other malignancies, characteristic mutations of β-catenin and APC leading to cytoplasmic stabilization of β-catenin were excluded by direct sequencing or protein truncation test. In patient tissues, β-catenin expression was directly and its degradation product's (β-catenin-P654) expression was inversely correlated with WHO grade. Increased β-catenin expression and low β-catenin-P654 expression were associated with shorter survival. Altogether, we report on potential canonical wnt pathway activation in high-grade gliomas and demonstrate that β-catenin expression in astrocytomas is associated with increased malignancy and adverse outcome.Aberrant  As crosrecently –4, this β-catenin, the main effector of the canonical  wnt pathway. In the absence of wnt ligands, β-catenin is regulated by a multiprotein complex comprising at least four interacting proteins: the tumour suppressor APC, the scaffolding protein axin, and the phosphokinases GSK-3β and CK1 [β-catenin accumulation with subsequent nuclear translocation. In the nucleus, β-catenin acts as an essential cofactor in the context of cell cycle regulation, differentiation, migration, proliferation, and survival [Activating mutations in this pathway contribute to neoplastic transformation in various human tissues. The common hallmark of these mutations is the nuclear accumulation of  and CK1 . Upon lisurvival .wnt pathway activation is mimicked by cytoplasmic β-catenin stabilization [β-catenin in high-grade gliomas leading to some transcriptional wnt activation. Concomitantly, we exclude characteristic activating mutations in the APC or β-catenin gene as a cause for cytoplasmic β-catenin stabilization, as they frequently occur in other tumour entities. Further, we describe an increased β-catenin expression in astrocytomas that correlates with malignancy grade and expression of the stemness marker nestin. In contrast, expression of β-catenin-P654, which can be regarded as inactivated degradation product of β-catenin [β-catenin and decreased β-catenin-P654 expression are associated with a poor patient outcome. Altogether, our data point to a potential canonical wnt pathway activation in astrocytic high-grade gliomas.In many cancers, among which colorectal cancers are probably the best studied example, aberrant lization –11. In t-catenin , decreasn = 16) were established as described [Short-term glioma cultures . Primers are summarized in Total RNA isolation from glioma cultures and RT-PCR reactions to determine mRNA expression of 2, 0.5% Triton X-100, 1x Complete Protease Inhibitor Cocktail , 20 μM DNAse I). The suspension was centrifuged for 10 min at 14.000 rpm and 4°C and the supernatant stored at −80°C. Protein concentration was quantified colorimetrically using the DC Protein Assay .For preparation of whole cell lysates, cells grown to 80% confluency were washed twice with PBS and lysed in CSK buffer . Following mechanical disruption of the cells, the lysate was centrifuged for 10 min at 600 g and 4°C, the pellet resuspended in homogenization buffer and centrifuged again. The collected supernatants of both centrifugation steps containing the combined membranous and cytoplasmic fraction were ultracentrifuged for 1 hour at 100.000 g and 4°C. The supernatant containing the cytoplasmic fraction was stored at −20°C. β-catenin  and HRP-coupled anti-mouse antibodies . Western Blot bands were quantified using the software ImageJ (http://rsbweb.nih.gov/ij/).Equal amounts of protein were separated on 10% polyacrylamide SDS gels, blotted onto PVDF Hybond P membranes , and detected with mouse anti-β-galactosidase expression plasmid pCMV-beta  to provide an internal control for the normalization of all data for transfection efficiency, cell viability, and cell number. pTOPFLASH contained the firefly luciferase ORF under control of a β-catenin/TCF regulated promoter. In the presence of endogenous nuclear β-catenin, luciferase transcription was activated by β-catenin/TCF. In pFOPFLASH, the TCF binding motif was defective. Cell lysis and luciferase and β-galactosidase detection were performed using the dual-light system  and the luminometer Lumat LB9507 . Cells were transfected using Effectene and Superfect transfection systems  following the manufacturer's instructions and harvested 24 h–48 h after transfection. Reporter plasmids  were a gift from H. Clevers . Cells were transfected with either pTOPFLASH or pFOPFLASH and cotransfected with the β-catenin exon 3 was amplified including exon-intron boundaries. Sequencing was performed using the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Kit . Sequencing products were resolved on an ABI 3100 automated sequencer  and analyzed using Staden Package [DNA was extracted from glioma cultures using the QiAmp DNA mini Kit  following the manufacturer's instructions. Codons 957 to 1513 of the APC coding sequence, containing the mutations cluster region as well as mutational hotspots at codon 1061 and codon 1309, were amplified .  Package .in vitro transcription. For in vitro translation, biotinylated lysine tRNA was used. The resulting protein fragments were separated by SDS-PAGE, transferred to a PVDF membrane, and detected using streptavidin-peroxidase. After reverse transcription of RNA extracted from glioma cells, APC cDNA was amplified in five segments. Segment 1 contained exons 2–15, segments 2–5 spanned the large exon 16. Each forward primer contained a T7 promoter, Kozak sequence, and start codon, to allow subsequent The tissue microarray (TMA) consisted of formalin-fixed, paraffin-embedded tissue samples derived from 283 patients operated at the Department of Neurosurgery at Heidelberg University, Germany, for astrocytomas of WHO grade II, III, or IV. Informed consent was obtained from each patient according to the research proposals approved by the Institutional Review Board at Heidelberg Medical Faculty. Median follow-up time was 11.0 years (±4.4 years). Overall survival was calculated from the first date of diagnosis till the time of death or the provisional endpoint of the study (November 30th 2009). Patient characteristics are reported in β-catenin  and mouse-monoclonal anti-β-catenin-P654 . Antigen retrieval was performed in a water bath at 100°C for 20 min using antigen retrieval buffers at pH 9.9 . Prior to TMA staining specificity of primary antibodies was ensured using appropriate isotype controls , and antigen expression was evaluated in a small study sample comprising astrocytoma tissues of WHO grades II–IV (n = 20 –30) to establish suitable antigen grading categories based on antigen expression variability . Staining was performed as previously described [β-catenin: 0% = 0; >0–10% = 1; >10–50% = 2; >50–80% = 3; >80–100% = 4; β-catenin-P654: 0% = 0; >0–1% = 1; >1–5% = 2; >5–10% = 3; >10% = 4. Average staining results from all biopsies of each individual patient were taken as final result. For statistical analysis, intermediate staining frequencies were used as cutoffs to group patients into “low expression” and “high expression” cohorts . For immunofluorescence staining, primary antibodies ; rabbit-polyclonal anti-nestin ) were visualized with fluorophore-conjugated secondary antibodies . 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining.Primary antibodies used for immunohistochemistry were mouse-monoclonal anti-escribed . Each biFor knockdown experiments, commercially available plasmids harboring CTNNB1 shRNA or a scrambled control were used following the manufacturer's instructions . Knockdown was performed on an additional cell line derived from a primary glioblastoma (NCH421k), which was chosen for its high expression of nestin .PITPNB) was used for internal normalization. The following primers were used in our study: CTNNB1 mRNA forward primer 5′-GCTTTCAGTTGAGCTGACCA-3′; CTNNB1 mRNA reverse primer 5′-CAAGTCCAAGATCAGCAGTCTC-3′; NESTIN mRNA forward primer 5′-TGCGGGCTACTGAAAAGTTC-3′; NESTIN mRNA reverse primer 5′-TGTAGGCCCTGTTTCTCCTG-3′; PITPNB forward primer 5′-CGAGACTCAGAAAGAACTAGAAACAA-3′; PITPNB reverse primer 5′-TGACCCTACAGGGGACTCAT-3′. RNA isolation was performed using the RNeasy Mini Kit  according to the manufacturer's instructions. An endogenous housekeeping gene (http://www.r-project.org/). P values ≤0.05 were considered statistically significant.Recurrent tumours and patients with incomplete clinical followup were excluded from the survival analysis. Association between overall survival (OS) and staining frequencies of individual antigens was calculated using the log-rank test and presented as Kaplan-Meier plots. In multivariate Cox regression analyses, hazard ratios were adjusted for the influence of prognostic factors on OS, that is, WHO grade, patient age at diagnosis, and extent of tumour resection. All calculations were performed using the statistical software environment R, version 2.4.1 , and one anaplastic oligodendroglioma WHO grade III (NCH262). The established glioblastoma cell lines A172 and U373 were included in the study as well as the human colon adenocarcinoma cell line SW480, which served as positive control.Presence and functional significance of wnt pathway was examined by RT-PCR  and in the two gliosarcoma cultures, whereas β-catenin was not present in the cytoplasm of the anaplastic oligodendroglioma analyzed (NCH262). Hence, the majority of our cultures featured an aberrant cytoplasmic β-catenin pool.Since aberrant Wnt/β-catenin signalling depends on the cytoplasmic stabilization of β-catenin. In order to exert its function as a transcription factor, however, β-catenin must first translocate to the nucleus. To investigate whether the aberrant cytoplasmic accumulation of β-catenin was accompanied by increased transcriptional activation we performed a luciferase reporter gene assay to directly assess transcriptional activity of the β-catenin/TCF complex. Glioma cultures were transiently transfected with reporter plasmids containing an intact TCF binding motif (pTOPFLASH) or a defective TCF binding motif (pFOPFLASH)  did not show transcriptional activity in the luciferase reporter gene assay. In the remaining 3/13 cultures only a borderline increase in reporter induction was measured despite cytoplasmic β-catenin accumulation. Altogether, there is evidence for wnt pathway activation by cytoplasmic stabilization of β-catenin in high-grade glioma, even though cytoplasmic accumulation was not necessarily succeeded by increased transcriptional activity in all glioma cell lines.OPFLASH) . 5/13 cunduction , all thewnt pathway have been described in various tumours [β-catenin binding site of APC. No APC mutations were found in any of the 13 glioma cultures that could have contributed to the accumulation of cytoplasmic β-catenin. Since ~95% of known APC mutations introduce a preterminal stop codon and lead to formation of a truncated protein, we further employed a protein truncation test to screen for additional APC mutations [in vitro. The size of the generated protein was then examined by SDS-PAGE  on a tissue microarray comprising samples of 283 astrocytomas of WHO grade II, III, or IV  showing the strongest expression in glioblastomas WHO grade IV  . Moreover, increased expression of β-catenin-P654 was associated with better OS . An adjustment for known prognostic confounders revealed that the association of β-catenin/β-catenin-P654 and survival were in part attributable to the relationship of immunoreactivity with WHO grade . Nevertheless, in astrocytomas of WHO grade II, we still found a significant association between increased expression of β-catenin-P654 and prolonged OS  . In astrβ-catenin is involved in tumor proliferation and/or tumor differentiation, we correlated the expression of β-catenin and β-catenin-P654 with a previously compiled data set generated on the same TMA. This included expression of the proliferation marker Ki-67 as well as the stem cell-associated intermediate filament protein nestin [β-catenin forms, we found a significant association between β-catenin and nestin expression in WHO grade IV tumours , which was confirmed by demonstrating colocalization of β-catenin and nestin by double immunofluorescence staining (β-catenin levels in vitro by shRNA did not affect nestin expression (β-catenin is not directly involved in the transcriptional regulation of nestin in our cell lines.Given the fact that, in other tumor entities, cytoplasmic accumulation of n nestin . Whereasstaining . Howeverpression , suggestwnt pathway activation through cytoplasmic stabilization of β-catenin is a key event in the development of numerous human malignancies. However, data on canonical wnt signalling in high-grade gliomas are sparse [β-catenin was identified as a frequent event in high-grade glioma cultures. In accordance with the common concept that cytoplasmic stabilization of β-catenin is the crucial step in regulating the canonical wnt signal, only tumors with a cytoplasmic pool of β-catenin showed increased transcriptional β-catenin/TCF-dependent activity in our study. However, presence of a cytoplasmic β-catenin pool did not always lead to transcriptional activity of β-catenin in the nucleus. Presumably, the balance of cytoplasmic versus nuclear retention factors of β-catenin as well as the presence of nuclear coactivators and corepressors might also modulate downstream β-catenin/TCF signalling in gliomas. Aberrant e sparse , 23. In wnt pathway mutations that cause cancer in various other tissues could be excluded as potential causes of cytoplasmic β-catenin accumulation in our study. This was shown by direct sequencing of the coding regions of APC and β-catenin and by an APC protein truncation test. The mechanism that causes cytoplasmic stabilization in gliomas, therefore, seems to be located upstream of the β-catenin degradation complex and might relate to downregulation of wnt pathway inhibitors or a shift of β-catenin from the membranous compartment to the cytoplasm by tyrosine phosphorylation of β-catenin. EGFR [β-catenin and thus activating wnt signalling. Additionally, signalling pathways that are commonly disinhibited in gliomas including the TGFβ/Akt and HGF pathway are also known to influence wnt signalling [wnt signalling, such as SFRP1, SFRP2, and NKD2, are often epigenetically silenced by promoter hypermethylation in glioblastomas of WHO grade IV and that these alterations occur independently of common β-catenin mutations [Interestingly, common in. EGFR , for insgnalling –4. Furthutations .wnt pathway activation and the clinical behavior of the respective tumor is well established. In human breast cancer, cytoplasmic and nuclear localization of β-catenin were shown to predict a poor outcome [β-catenin is established as a favorable-risk indicator in a subgroup of medulloblastomas [wnt signalling. Here we report on a WHO grade-dependent increase in β-catenin and decrease in the inactivated β-catenin-P654 in a large study sample. β-catenin-P654 expression even was significantly associated with prolonged OS. These results are supported by a recent study that demonstrated a significant increase of β-catenin mRNA expression levels in high-grade astrocytomas compared to low-grade astrocytomas and normal brain tissue [β-catenin and the neural stemcell-associated protein nestin. Although our study suggests that β-catenin is not directly involved in the transcriptional regulation of nestin, wnt/β-catenin signalling could be involved in maintaining an undifferentiated, stem-like glioma cell phenotype. Consistently, the canonical wnt pathway has been reported to modulate the balance between stemness and differentiation in several adult stem cell niches [For various human cancers, an association between aberrant  outcome , 27 wherlastomas . In human tissue . Finallyl niches  includinl niches –33. β-catenin in astrocytic gliomas is linked to a higher tumor grade and coincides with the expression of the stemness marker nestin. However, an impact on patient outcome was especially observed in low-grade gliomas.Taken together, our results imply that aberrant expression of