H2 cytokines in Wnt-dependent manner and orchestrate TH2 lineage commitment.Chromatin organizer SATB1 and Wnt transducer β-catenin form a complex and regulate expression of GATA3 and T H2) specific transcription factor that regulates production of TH2 cytokines and functions as TH2 lineage determinant. SATB1 positively regulated GATA-3 and siRNA-mediated knockdown of SATB1 downregulated GATA-3 expression in differentiating human CD4+ T cells, suggesting that SATB1 influences TH2 lineage commitment by reprogramming gene expression. In the presence of Dickkopf 1 (Dkk1), an inhibitor of Wnt signalling, GATA-3 is downregulated and the expression of signature TH2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that Wnt signalling is essential for TH2 differentiation. Knockdown of β-catenin also produced similar results, confirming the role of Wnt/β-catenin signalling in TH2 differentiation. Furthermore, chromatin immunoprecipitation analysis revealed that SATB1 recruits β-catenin and p300 acetyltransferase on GATA-3 promoter in differentiating TH2 cells in a Wnt-dependent manner. SATB1 coordinates TH2 lineage commitment by reprogramming gene expression. The SATB1:β-catenin complex activates a number of SATB1 regulated genes, and hence this study has potential to find novel Wnt responsive genes. These results demonstrate that SATB1 orchestrates TH2 lineage commitment by mediating Wnt/β-catenin signalling. This report identifies a new global transcription factor involved in β-catenin signalling that may play a major role in dictating the functional outcomes of this signalling pathway during development, differentiation, and tumorigenesis.In vertebrates, the conserved Wnt signalling cascade promotes the stabilization and nuclear accumulation of β-catenin, which then associates with the lymphoid enhancer factor/T cell factor proteins (LEF/TCFs) to activate target genes. Wnt/β -catenin signalling is essential for T cell development and differentiation. Here we show that special AT-rich binding protein 1 (SATB1), the T lineage-enriched chromatin organizer and global regulator, interacts with β-catenin and recruits it to SATB1's genomic binding sites. Gene expression profiling revealed that the genes repressed by SATB1 are upregulated upon Wnt signalling. Competition between SATB1 and TCF affects the transcription of TCF-regulated genes upon β-catenin signalling. GATA-3 is a T helper type 2  are required for differentiation of Th2 cells, an important subset of helper T cells. Specifically we show that siRNA-mediated knockdown of SATB1 downregulated  Wnt growth factors regulate a variety of developmental processes by altering specific gene expression patterns −CD8− (DN), CD4+CD8+ (DP), and CD4+CD3+ or CD8+CD3+ (SP). In addition to the well-studied T cell receptor (TCR)-mediated signals and the Notch pathway, thymic epithelial cells also contribute towards thymocyte development and differentiation by producing Wnt H) cells. Upon antigenic stimulus, naïve CD4+ TH cells differentiate into distinct effector cell lineages referred to as TH1 and TH2. This differentiation is mediated by signalling proteins such as STAT4 and STAT6 and is accompanied by upregulation of marker transcription factors such as GATA-3 and c-Maf in TH2 cells and T-bet in TH1 cells + T cells into TH2 and TH1 cell types, respectively H cell differentiation have been studied extensively, there is no report demonstrating the direct role of Wnt signalling in TH cell differentiation.Within the thymus, thymocyte maturation involves a series of discrete phenotypic stages that correspond to developmental checkpoints and are subsequently referred to as CD4H2) cytokine and MHC class-I loci into distinct chromatin loops by tethering MARs to the nuclear matrix at fixed distances H2 cytokine genes H cells c-MycBcl-2The T-lineage-enriched chromatin organizer special AT-rich sequence binding protein 1 (SATB1) was shown to regulate distant genes by selectively tethering matrix attachment regions (MARs) to the nuclear matrix H2 lineage commitment via Wnt/β-catenin signalling. As a functional consequence of abrogating function of SATB1 or β-catenin or Wnt signalling, we demonstrate that the production of signature TH2 cytokines such as IL-4, IL-10, and IL-13 is reduced, indicating that TH2 differentiation is affected. Thus, these studies establish SATB1 as a determinant of TH2 differentiation and downstream effector in the Wnt/β-catenin signalling pathway.In this study, we set out to unravel the molecular mechanisms contributing towards the functional overlap between SATB1 and β-catenin pathways, especially with respect to their target gene regulation. We show that SATB1 physically interacts with β-catenin and recruits it to its genomic targets. Interestingly, deacetylation of SATB1 upon Wnt/β-catenin signalling leads to an increase in its occupancy on genomic targets. Recruitment of β-catenin alters the transcription of SATB1's target genes in thymocytes. Chromatin immunoprecipitation (ChIP) analysis of SATB1 binding sites (SBSs) in promoters of multiple genes revealed that interaction with β-catenin modulated SATB1 function on its target genes by increasing its occupancy and altering histone H3 lysine 9 (H3K9) acetylation. β-catenin-responsive genes are also targeted by SATB1, suggesting that both are functionally linked in the Wnt/β-catenin signalling pathway. Additionally, β-catenin-induced TCF responsive genes are dysregulated upon SATB1 overexpression, suggesting competitive interaction of TCF/LEF and SATB1 with β-catenin. Furthermore, SATB1 directly regulates GATA-3 expression and dictates TGene expression profiles of cells expressing phosphorylation- or acetylation-defective mutants of SATB1 BCL-2CHUK, PPM1A, c-Myc, and IL-2FOSB and LBCL-XThPOK (ZBTB1-7B) showed a reciprocal pattern of regulation. ThPOK was downregulated upon Wnt3a treatment and dramatically upregulated (more than 20-fold) upon inhibition of Wnt signalling by Dkk1. Among the known Wnt targets in thymocytes, few genes such as TRAIL and PP2ATIMP1 that is neither targeted by SATB1 nor targeted by β-catenin is not affected by Wnt3a or Dkk1 treatment of thymocytes, indicating that this effect is specific to SATB1 and Wnt/β-catenin targets  consisted of aa 1–576 and only C-terminus harbouring construct that of aa 577–780. We tested the possibility of whether overexpression of this interaction region works as a dominant negative for SATB1 and β-catenin interaction. Towards this, we overexpressed 1–576 and 577–780 regions of β-catenin as red fluorescent protein (RFP) fusions in Human Embryonic Kidney (HEK)-293T cells and induced β-catenin signalling by adding LiCl. SATB1 immunoprecipitation followed by immunoblot analysis with anti-β-catenin confirmed that they form a complex in LiCl-treated cells , lane 1.B1 c-Myc . Thus, oart site . Next, w-Myc SBS , bar 6. -Myc SBS , bar 5. -Myc SBS , providi-Myc SBS , overexp-Myc SBS , bar 8. Since we tested transcription of a number of genes that are targets of both SATB1 and TCF-1, we tested whether they interact with each other. Immunoprecipitation of thymocyte extract with anti-TCF-1 did not pulldown SATB1, suggesting a lack of their physical interaction , lane 2.IL-2 promoter-mediated reporter activity was upregulated in a time-dependent manner upon LiCl treatment . Treatment with BIO resulted in stabilization of β-catenin predominantly in TH2 cells  H2 cells were cultured. Cells were differentiated for 72 h and supplemented with Dkk1 after every 24 h. Expression of GATA-3 was monitored as an indicator of the TH2 differentiation. Quantitative transcript profiling revealed that GATA-3 expression was suppressed by over 3-fold upon Dkk1 treatment in TH2 subset, suggesting that Wnt signalling is necessary for the upregulation of GATA-3 during differentiation of TH2 cells . Notably, in TH2 cells GATA-3 was downregulated more than 11-fold upon SATB1 knockdown (bar 6). The downregulation of GATA-3 in TH2 was more striking as compared to the TH0 subset, indicating that SATB1 is required for GATA-3 expression in TH2 cells. The influence of SATB1 on GATA-3 expression was further confirmed by overexpression of SATB1 that led to a significant increase in the expression of GATA-3 in both subsets as compared to the respective controls , suggesting that SATB1 positively regulates GATA-3 expression. Under these conditions, the expression of another TH2-specific transcription factor c-Maf was not altered significantly  served as the control. Both siSATB1 and Scr transfected cells were differentiated into TH2 cell lineage by adding IL-4 over the next 72 h. Cells that were only activated by plate bound anti-CD3 and anti-CD28 and not treated with IL-4 were referred to as TH0. We subjected these differentiated cells to ChIP analysis and monitored the occupancy of SATB1 and β-catenin at the SBS within the upstream region of GATA-3 promoter by quantitative PCR analysis of the eluted chromatin. As a control for the ChIP analysis, we used another region at −1,500 to −1,800 bp of GATA-3 promoter that was not directly bound by SATB1 (non-SBS) . Strikingly, β-catenin occupancy also increased during the differentiation of control TH2 cells from 24 to 72 h and was significantly reduced upon SATB1 knockdown . Such increase in the occupancy of SATB1 or β-catenin was not observed at the upstream non-SBS during the time course of differentiation (bars 21–32), suggesting that SATB1 recruits β-catenin at the SBS in GATA-3 promoter.To understand whether SATB1 and β-catenin regulate  by EMSA . Next, wnon-SBS) . The occGATA-3 promoter, we inhibited the Wnt signalling by treating the differentiating TH cells with Dkk1. The TH cells were differentiated as mentioned above and treated with Dkk1 for 72 h. The activated TH cells (TH0) were used as controls. We differentiated cells for 72 h because the maximum occupancy of β-catenin and SATB1 on GATA-3 was observed upon 72 h of differentiation. Chromatin isolated from differentiated TH0 (control) and TH2, Dkk1 treated, and untreated cells was subjected to immunoprecipitation using antibodies to SATB1, β-catenin, and p300. The occupancy of these proteins at the SBS within the GATA-3 promoter was monitored during Wnt-on and -off conditions by quantitative PCR of immunoprecipitated DNAs. Upon Dkk1 treatment (Wnt-off), since the nuclear β-catenin level is reduced, its occupancy on GATA-3 promoter was also reduced significantly (GATA-3 promoter upon Dkk1 treatment (compare bars 11 and 12), corroborating the observation that the SATB1:β-catenin complex recruits p300. This further explains the observed downregulation of GATA-3 upon Dkk1 treatment . SATB1 presumably contributes to all of the above since in SATB1 null mice the development of thymocytes is arrested at the DP stage, very few CD8 SP cells are observed SATB1 is upregulated early during TH cell differentiation H cell development, and the role of Wnt signalling in this process is also unprecedented. We show that CD4+ T cells are receptive to Wnt signals presumably because they produce different Wnts themselves. The differential sensitivity of TH cell subtypes to Wnt signalling could be due to the fact that the downstream processes such as stabilization of β-catenin occur prominently in the TH2 subtype. Our study defines the role of SATB1:β-catenin collaboration in this important biological phenomenon. Further, SATB1 reprogrammes chromatin organization and gene expression profiles to promote breast tumor growth and metastasis A number of studies have specifically addressed the relevance of Wnt signalling for lymphocyte development and typically involve genetic and/or biochemical analyses of TCF functions Pit1 and represses the gene encoding the lineage-inhibiting transcription factor Hesx1 acting via the TLE/Reptin/HDAC1 corepressor complexes In light of our findings, we propose that in absence of Wnt signalling when β-catenin is phosphorylated and degraded, SATB1 acts predominantly as a repressor on most of the genes and recruits different chromatin remodelling complexes to its genomic targets. However, upon β-catenin stabilization, it associates with β-catenin inside the euchromatic nuclear compartment, and its binding to target DNA is enhanced along with recruitment of β-catenin and H3K9 acetylation. Thus our study provides a mechanism explaining how SATB1's regulatory functions are modulated from repressor to activator in a signal-dependent manner. SATB1 acts as a “landing platform” for chromatin remodelling factors Deacetylation of SATB1 upon Wnt signalling is a key event modulating transcription. Acetylation has been shown to abrogate the DNA-binding activity of SATB1 H2 specific cytokine locus has been suggested wherein SATB1 facilitates the expression of IL-4, IL-5, IL-13, and c-Maf via formation of a densely looped, transcriptionally active, higher-order chromatin structure upon TH2 cell activation H cell differentiation is mediated by signalling proteins, including STAT4 and STAT6, resulting in expression of transcription factors such as GATA-3 and c-Maf (in TH2 cells) or T-bet (in TH1 cells) H2 responses through three different mechanisms: induction of TH2 cytokine production, selective growth of TH2 cells, and inhibition of TH1 promoting factors H2 polarization H2 cells and SATB1 is also upregulated in TH2 cells H cell differentiation, we silenced SATB1 expression and monitored the TH cell differentiation by quantifying GATA-3 expression. Interestingly, upon siRNA mediated knockdown of SATB1, GATA-3 expression is drastically decreased, indicating that TH2 polarization is SATB1-dependent. This is further substantiated by results of SATB1 overexpression in TH2 cells, which clearly establish SATB1 as a positive regulator of GATA-3. The Wnt/β-catenin signalling is active in CD4+ TH cells as revealed by stabilization of β-catenin. The differential stabilization of β-catenin in TH1 and TH2 subtypes may explain the differential pattern of gene expression in these cells. We therefore used Dkk1 treatment to inhibit the active Wnt signalling in TH2 cells. Upon Dkk1 treatment, GATA-3 expression was reduced drastically, suggesting that GATA-3 is regulated in a Wnt/β-catenin signalling-dependent manner. Notably, c-Maf is not regulated by SATB1 or β-catenin under these conditions. Thus, at least during the early commitment and differentiation of human CD4+ TH cells, SATB1 does not seem to regulate c-Maf. SATB1 regulates GATA-3 by directly binding to its promoter elements and may not regulate c-Maf in this manner. However, further studies may be required to address the role of SATB1 in regulation of c-Maf during different stages of TH differentiation since positive regulation of c-Maf by SATB1 has been demonstrated in the differentiated mouse TH2 cell line (clone D10) H2 cytokines such as IL-4, IL-10, and IL-13 is also positively regulated by both SATB1 and Wnt signalling, as evident from their downregulation upon silencing of SATB1 or β-catenin and inhibition of Wnt signalling by Dkk1. The early time points in differentiation as employed in this study do not yield a very high amount of secreted cytokines in cell culture supernatants. Interestingly, at the RNA level as well as at the intracellular protein level, dramatic downregulation of IL-4 was observed. The secretion of IL-4 and other cytokines presumably surges after 72 h of differentiation. However, the silencing effect of siRNA would not last over such a long time period, and therefore effects of siSATB1 or siβ-catenin could not be monitored beyond 72 h in culture. Intriguingly, IL-10 gene is located outside of the TH2 cytokine locus and yet is co-ordinately regulated by SATB1 in a Wnt-dependent manner. In GATA-3-deficient TH2 cells, production of signature TH2 cytokines such as IL-4 and IL-13 as well as that of IL-10 was also reduced, indicating that GATA-3 is a major orchestrator of coordinated TH2 response GATA-3 induced TH2 cytokine expression in both differentiating and irreversibly committed TH1 cells GATA-3 expression directly affects TH2 differentiation. Thus, in the presence of the inhibitor of Wnt signalling (Dkk1) or upon knockdown of β-catenin, TH2 differentiation seems to be affected due to the downregulation of GATA-3. We therefore propose that SATB1 mediates TH2 differentiation by regulating GATA-3 expression and thereby the expression of signature TH2 cytokines in a Wnt/β-catenin-dependent manner. Interestingly, a recent study reported that nephric duct-specific inactivation of GATA-3 leads to massive ectopic ureter budding, suggesting that GATA-3 acts downstream of β-Catenin signalling to prevent ectopic metanephric kidney induction H2 differentiation of activated CD4+ T cells by promoting GATA-3 expression and suppressing IFN-γ expression GATA-3 is regulated by Wnt signalling.Role of SATB1 in regulation of TGATA-3 expression by the SATB1:β-catenin complex in a Wnt signalling-dependent manner. ChIP analysis of an SBS within the GATA-3 promoter in differentiating TH2 cells revealed a time-dependent increase in occupancy of SATB1 and β-catenin. Upon silencing of SATB1, its occupancy was expectedly decreased, but remarkably, even that of β-catenin was decreased by several-fold, suggesting that SATB1 is the key factor mediating the recruitment of β-catenin on GATA-3 and presumably other targets. The recruitment of the SATB1:β-catenin complex on GATA-3 promoter is sensitive to Dkk1 treatment. Moreover, since β-catenin recruits p300 on SATB1 genomic targets, the occupancy of p300 is also reduced concomitantly as that of β-catenin, presumably leading to the observed downregulation of GATA-3 upon inhibition of Wnt signalling by Dkk1 treatment. Although control TH0 cells also show a similar Dkk1 sensitive pattern of occupancy of these three factors on the GATA-3 promoter SBS, their fold occupancy is significantly more in the TH2 cells. The occupancy of the SATB1:β-catenin complex is very low at a non-SBS located upstream on the GATA-3 promoter and is not sensitive to Dkk1. As with multiple other genes, the differential occupancy of various factors on different regions of the same promoter seems to be the mechanism governing regulation of transcription for GATA-3. The intriguing observation is that such differential recruitment is dependent on Wnt signalling. Thus, our results provide a molecular mechanism towards understanding the role of Wnt signalling in TH2 differentiation and define a unique role of SATB1 in this process.Our study also provides a molecular mechanism for regulation of Antibodies to β-catenin and SATB1 were purchased from BD Biosciences. Antibodies to PCAF and pan-acetyl lysine were from Santa Cruz Biotechnology; antibodies to N-terminus of β-catenin, VP-16, and H3K9ac were obtained from Upstate Technologies. Anti-Flag was procured from Sigma-Aldrich Corp. siSATB1 and siβ-catenin duplex RNAs were from Santa Cruz Biotechnology, and BIO was obtained from Calbiochem. Recombinant Wnt3a, Dkk1, and cytokines were obtained from R&D Systems. Anti-SATB1 used for IP and WB was raised in rabbit and was purified using immunoaffinity chromatography using standard procedures.Expression constructs for β-catenin mutants T41A and GST 1–12 arm repeats were kindly provided by Dr. C. Neuveut, and pGEX6P3-β-catenin was gifted by Dr. M. Dunach. Deletion construct pGEX6P3-β-catenin-1–535 was generated by digesting pGEX6P3-β-catenin with SpeI and XhoI and relegating the larger fragment into pGEX6P3. pGEX6P3-β-catenin was digested with EcoRI, and the 1 Kb fragment was subcloned into pGEX6P2 to obtain pGEX6P2-β-catenin-425–781. RFP-fused versions of truncated β-catenin were produced by subcloning regions of β-catenin cDNA corresponding to aa 1–576 and 577–780 in pDsRed Express C1 vector (Takara Clontech). Various VP16-β-catenin truncations and Gal4DBD:SATB1, and Gal4DBD:PDZ fusions were cloned in pACT and pBIND plasmids (Promega), respectively. TOP and FOP reporter constructs were kind gifts by Dr. R. T. Moon. pTriEx-SATB1, various SATB1 domain constructs, and the reporter constructs pGL3-Basic-IgH MAR and pGL3-Basic-IL-2 have been described previously Thymocytes were isolated from 3-wk-old Balb/C mice. Thymocytes were fixed using 2% paraformaldehyde and were permeabilized with 0.1% Triton X-100 followed by antibody staining of SATB1 and β-catenin (BD Biosciences). The secondary antibodies used were conjugated to Alexafluor dyes 488 and 594 (Invitrogen). DNA counterstaining was performed using DAPI. Cells were visualized under an upright fluorescence microscope , and digital images were enhanced using the Apotome module (Carl Zeiss). The sectional views of the stained cells revealing the signals from the nuclear interior were generated using the Axiovision software (Carl Zeiss).Escherichia coli (E. coli). Specific fusion proteins were cleaved on column by caspase-6 to obtain GST-free proteins as described 35S-labeled SATB1 was prepared by coupled in vitro transcription and translation as per manufacturer's instructions (Promega). Full-length SATB1 was expressed as 6XHis-SATB1 in E. coli and was bound to Ni-NTA beads (Qiagen). Bound proteins were washed with 1× PBS and incubated either with affinity purified recombinant proteins or with 100 µg nuclear extract from Jurkat or SW 480 cells or with in vitro transcribed and translated SATB1 for 2–3 h. The complexes were then washed with 1× PBS containing 0.1% triton X-100 and eluted using 2× SDS-loading buffer, resolved by SDS-polyacrylamide gel electrophoresis and analyzed by immunoblotting.GST:1–178, GST:1–535, GST:425–781, and GST:Arm repeats of β-catenin and GST:PDZ, GST:CD+HD, GST:CD, and GST:HD of SATB1 were expressed and purified from + T cells were cotransfected with the indicated expression vectors and the TOPFlash or FOPFlash reporter constructs. Equal masses of DNA were used in each transfection, and vector DNAs were used for normalization of DNA if required in each transfection reaction. The ratio of luciferase activities in TOPFlash-transfected versus FOPFlash-transfected cells was calculated and plotted as the relative TCF activity. All reporter assays were repeated at least thrice using independently transfected HEK 293T cells unless mentioned otherwise, and bar graphs represent values +/− s.d. The statistical significance of observed differences was calculated by t test.HEK cell line 293T, colon cancer cell line SW480, and T cell lymphoblastoid Jurkat cells were cultured in DMEM or in RPMI 1640 (Invitrogen), respectively, supplemented with 10% FCS (Invitrogen). Cells were transfected with indicated expression constructs using Lipofectamine 2000 (Invitrogen). After transfections cells were cultured for 24 h and then either LiCl or BIO were added to media at final concentrations of 20 mM or 1.0 µM, respectively, and cells were cultured further for 24 h unless mentioned otherwise. Transactivation assays were performed after 48 h of transfections as described Five hundred µg of nuclear extract was diluted three times using 1× PBS containing 0.1% Triton X-100 and was precleared using 1.0 µg of either rabbit or mouse IgG and protein A/G beads (Pierce). Precleared lysate was subjected to immunoprecipitation by incubating with anti-SATB1 or anti-β-catenin for 2 h; protein A/G beads were then added and mixed further by incubating on an end-to-end rotator at 4°C for 4 h. Protein complexes were analyzed by immunoblotting with respective antibodies.ChIP was performed essentially as described Target−Ct Input). Fold differences in gene expression were calculated as follows: Fold difference = 2 (Treatment−Loading Control)/2 (Control−Loading Control). Fold changes were calculated by dividing the level of expression of the experimental sample with the corresponding control sample.Quantitative PCRs were performed using iCycler (BioRad) using iQ SYBR Green mix (Bio Rad). ΔCt values were calculated using the formula: ΔCt =  as per manufacturer's instructions.Peripheral human mononuclear cells were isolated from the human umbilical cord blood procured from local hospitals using Ficoll-Paque . CD4+ T cells were cultured in RPMI 1640 media supplemented with 10% FCS (Invitrogen). Cells were cultured in 24 well plates precoated with 0.5 µg/ml of anti-CD3 and in the presence of 0.5 µg/ml soluble anti-CD28 (eBiosciences). TH1 polarization was induced using 2.5 ng/ml IL-12 and TH2 polarization with 10 ng/ml IL-4 (R&D Systems). After 48 h of polarization, 17 ng/ml of IL-2 (R&D Systems) was added. Polarized cells were grown further for various time points from 24 to 72 h.CD4H cells were collected by centrifugation. Levels of various cytokines in these supernatants were measured using the Bio-Plex TH1-TH2 kit and the Bio-Plex protein array reader (Bio-Rad) as per manufacturer's instructions. Levels of secreted cytokines were normalized against the respective media.Supernatants from the control and treated T+ T cells were electroporated using the Nucleofector device (Amaxa) according to manufacturer's instructions. Cells were cultured in RPMI 1640 media supplemented with 10% FCS for 24 h. After 24 h cells were activated and polarized as described above. Wnt signalling was induced by addition of purified recombinant Wnt3a (R&D Systems) at 50 ng/ml or inhibited by addition of Dkk1 (R&D Systems) at 400 ng/ml. After 72 h of polarization in presence or absence of Dkk1, cells were harvested and divided into three parts. Chromatin was prepared from two portions of cells as described above. Total RNA was isolated from the remaining one third portion of cells, and cDNA was prepared using the Cells-to-Signal kit (Ambion) as per the manufacturer's instructions.Isolated T cells were differentiated ex vivo as described Figure S1Common targets of SATB1 and β-catenin. Expression profiling of cells in which β-catenin was overexpressed, knocked out, or mutated available on NCBI public domain GEO datasets GSE1473 p value (<0.05) are tabulated. Description of microarray datasets used: The data from datasets GSE1473, GSE2519, and GSE1579 are in Affymetrix format, whereas the dataset GSE4317 is in UHN cDNA array format. Data deposited in GSE4317 were generated in our lab and contain gene expression profiling data from Jurkat (T-cell) and HeLa (non-T cell), where the cDNA from control untransfected cells was referenced and cells overexpressing wild-type SATB1, SATB1-phosphorylation deficient S185A mutant, and SATB1-acetylation deficient K136A mutant were the “treated” cells. GSE1473 represents expression profiling of 293T cells infected with RCAS vector carrying β-catenin S37A mutant. β-catenin S37A mutant is oncogenic and more stable than the corresponding wild-type protein. GSE1579 contains analysis of skin of deltaNβ-cateninER transgenics following the activation of β-catenin for up to 7 d. Onset and duration of β-catenin activation in deltaNβ-cateninER transgenics was controlled by 4-hydroxytamoxifen. Results provide insight into how β-catenin induces hair follicle growth. Method for analysis of microarray data: The data from these four datasets were downloaded from GEO (www.ncbi.nlm.nih.gov/GEO). We used the RAW scores and normalized these scores with controls to obtain fold-increase/decrease. For the datasets where replicate samples were available, we used sample means to calculate the p-value for the two-tailed Student's t test. For other datasets we used the probe-means to calculate the p-value. In study for dataset GSE 1473 above, the p-values are for the genes affected because of constitutive expression of the S37A in the transfected cells and are also SATB1 targets. In study for dataset GSE 1579 above, the p-value is for the genes that are differentially expressed on day 7 as compared to day 0 and are SATB1 targets based on GSE 4317.(0.05 MB TIF)Click here for additional data file.Figure S2SATB1 and β-catenin interact in vivo. (A) Coimmunoprecipitation reactions was performed by separately incubating anti-β-catenin and anti-SATB1 antibodies with aliquots of Jurkat nuclear extract as described in (1.58 MB TIF)Click here for additional data file.Figure S3Qualitative analysis of proteins used for in vitro pulldowns. Coomassie stained gels depicting the purity of various GST-β-catenin  and SATB1  truncations used in GST pulldown experiments. His-SATB1 corresponds to 6× histidine-tagged full-length SATB1, CD+HD is the C-terminal half of SATB1 containing the DNA-binding region, whereas PDZ indicates the N-terminal 1–204 amino acids of SATB1 harbouring its PDZ-like signalling domain.(0.24 MB TIF)Click here for additional data file.Figure S4β-catenin activates SATB1-mediated transcription. The functional consequence of overexpression of SATB1 and constitutively active mutant β-catenin (T41A) on MAR-linked reporter assay was monitored as described in (0.10 MB TIF)Click here for additional data file.Figure S5Human thymocytes were isolated from neonatal thymii as described in . In brief, thymocytes were cultured on monolayers of OP9 or OP9-DL1 cells for 48 h in RPMI 1640 medium (Invitrogen) supplemented with 10% FCS, along with Wnt3a (10 µg/ml) (Wnt agonist) or DKK1 (100 ng/ml) (Wnt inhibitor). The co-cultures were also supplemented with rIL-7 (5 ng/ml) and FlT3 ligand (5 ng/ml) (R&D Systems). (A) The cells were harvested after 48 h and the cell viability was monitored by MTT assay using standard protocol. Briefly, Thiazolyl Blue Tetrazolium Bromide (USB) was added to the thymocyte culture at a concentration of 1 mg/ml and incubated for 1 h. Cells were harvested by centrifugation, and cell pellet was resuspended in DMSO and measured at 570 nm. The absorbance readings were converted to percentages in viability, and the viability of control untreated cells was normalized to 100%. (B) Cell death was also assessed by the trypan blue dye exclusion method after 48 h co-culture. Thymocytes were harvested after respective time points and resuspended in PBS. To the cell suspension, few drops of 0.4% Tryan blue were added and incubated for 5 min at room temperature. Cells were then observed using hemocytometer, and cells that have not internalized the dye due to active dye exclusion were counted and converted to percentage viability. The viability of control untreated cells was normalized to 100%. As shown in (A) and (B), no significant difference was observed in the viability of thymocytes treated with Wnt3a or DKK1 and co-cultured with control OP9 or OP9-DL1 cells.  Effect of Wnt signalling on the transcription status of representative Wnt regulated genes BCL-XL (C) and PP2A (D) in thymocytes. Thymocytes were co-cultured with control OP9 or OP9-DL1 cells as described above. Quantitative RT-PCR analysis was performed using RNA extracted from control human thymocytes and thymocytes treated for 48 h with Wnt3a or Dkk1 as described in (0.39 MB TIF)Click here for additional data file.Figure S6Analysis of SBSs in regulatory regions of multiple genes. EMSA analysis was performed using 32P-labeled upstream regulatory regions from CHUK, PPM1A, c-Myc genes, and the major breakpoint region of BCL2. Purified GST was used as a control for the GST:SATB1 fusion protein. The multiple regions tested from the 1 kb upstream region of CHUK and PPM1A are depicted schematically on top of the EMSA panels. Two regions  bound SATB1 at very high concentrations indicating very low affinity. Stars indicate SBSs whereas circles denote non-binding regions. Panel on bottom left depicts the dose-dependent binding of recombinant SATB1 with a 32P-labeled region encompassing −1,183 to −1,383 bp of human c-Myc promoter.(0.72 MB TIF)Click here for additional data file.Figure S7SATB1 and β-catenin occupy the SBSs in IL-2 and c-Myc promoters in vivo. (A) ChIP-PCR analysis in Jurkat cells showing the binding of SATB1 and β-catenin on IL-2 promoter in presence (+) and absence (−) of LiCl. ChIP analysis was performed as described in c-Myc promoter under these conditions . An upstream region (−10 kb) of c-Myc was used as a negative control for ChIP (lower panel).(0.11 MB TIF)Click here for additional data file.Figure S8SATB1 does not bind to the TCF consensus in vitro. In vitro binding analysis of TCF consensus binding site using recombinant SATB1 was performed by EMSA as described in 32P-labeled wild-type (lanes 1–5) and mutant TCF (lanes 6–10) consensus sequences. 32P-labeled IgH-MAR was used as a positive control for SATB1 binding (lanes 11–15).(0.18 MB TIF)Click here for additional data file.Figure S9+ T cells to TH1 and TH2 subsets.Confirmation of polarization of CD4 Quantitation of marker cytokines in culture supernatants harvested from TH cells grown for 72 h was performed simultaneously using a multiplex bead array reader as described in H2 cells produced IL-4, confirming that the cells were committed to the respective lineages.(5.36 MB TIF)Click here for additional data file.Figure S10H cells.Analysis of c-Maf expression in differentiating T Naïve CD4+ T cells were transfected with duplex siSATB1, siβ-catenin, or SATB1 overexpression plasmid DNA and differentiated ex vivo as described in GATA-3 transcripts were analyzed by quantitative RT-PCR as described in H0 (bars 1–4) and TH2 (bars 5–8) cells. Changes in expression levels were calculated with respect to the scrambled RNA transfected TH0 subset in which the c-Maf expression level was set to 1 (bar 1). Each error bar represents standard deviation calculated from triplicates. Naïve CD4+ T cells were transfected with duplex siSATB1, siβ-catenin, or SATB1 overexpression plasmid DNA as described in GATA-3 transcripts were analyzed by quantitative RT-PCR as described in GATA-3 expression was normalized with β-actin expression in these cells. The graph shows fold changes in GATA-3 transcript in control (Scr) (bar 1), SATB1 silenced (bar 2), SATB1 overexpressed (bar 3), and β-catenin silenced (bar 4) TH2 cells. Fold changes were calculated with respect to the scrambled RNA transfected TH0 subset in which the GATA-3 expression level was set to baseline (bar 1). Each error bar represents standard deviation calculated from triplicates.(0.11 MB TIF)Click here for additional data file.Figure S11H2 cells.IL-4 is downregulated upon siRNA-mediated silencing of SATB1 in differentiating T Human CD4+ T cells were transfected with Scrambled (Scr) and si SATB1 synthetic duplex RNAs and then polarized to TH2 as described in + T cells were polarized under TH2 conditions and harvested after 3 d. Four h prior to harvesting the cells, Brefeldin A was added to the culture media. The cells were fixed using 1% paraformaldehyde for 20 min at room temperature. The cells were then permeabilized using 0.5% Saponin and stained with anti-IL-4-PE conjugate (BD Biosciences) for 30 min. Stained cells were acquired on flow cytometer  and analyzed using CellQuest software (BD Biosciences).(1.96 MB TIF)Click here for additional data file.Figure S12Analysis of upstream regulatory regions of GATA-3. In vitro binding analysis of GATA-3 promoter using recombinant SATB1 was performed by EMSA as described in GATA-3 was monitored by using 32P-labeled fragments of this region. We focused on a high affinity SBS at a region spanning −900 to −600 bp and a non-SBS at an upstream region encompassing −1,800 to −1,500 bp of the human GATA-3 promoter. The schematic on top of the EMSA panels depicts the relative positions of these regions within the promoter. A star indicates position of SBS, whereas a circle denotes non-binding site.(0.25 MB TIF)Click here for additional data file.Table S1List of genes and gene IDs. The genes studied here or mentioned in the text are tabulated along with their respective gene IDs.(0.05 MB DOC)Click here for additional data file.Video S1SATB1 and β-catenin colocalize in the nuclei of mouse thymocytes. Triple stained immunofluorescence image . Thymocyte nuclei were counterstained with DAPI to mark the DAPI intense and less intense areas in the nuclei. The movie depicts a 3-D stack of images from top to bottom in the same thymocytes as in (0.05 MB WMV)Click here for additional data file.Video S2β-catenin forms a characteristic “cage-like” substructure in the nuclei of mouse thymocytes. Single stained immunofluorescence image (Alexa 488). The movie depicts a 3-D stack of images from top to bottom in the same thymocytes as in (0.04 MB WMV)Click here for additional data file.


The 249I and 280M alleles were             associated with PAH . In conclusion,             the increased frequencies of 249I and 280M CX3CR1 alleles in a subgroup of             patients with SSc-associated PAH suggest a role for the fractalkine system in             the pathogenesis of this             condition. Further, the 249I allele might be associated with susceptibility to SSc.Fractalkine (FKN) and its receptor CX3CR1 are critical mediators in the             vascular and tissue damage of several chronic diseases, including systemic             sclerosis (SSc) and pulmonary arterial hypertension (PAH). Interestingly, the V249I             and T280M genetic polymorphisms influence CX3CR1 expression and function. We             investigated whether these polymorphisms are associated with PAH secondary to             SSc. CX3CR1 genotypes were analyzed by PCR and sequencing in 76 patients with             limited SSc and 204 healthy controls. PAH was defined by colorDoppler echocardiography.             Homozygosity for 249II as well as the combined presence of 249II and 280MM were             significantly more frequent in patients with SSc compared to controls (17 vs 6%,


ΔN89β-catenin activated signaling within a luminal subpopulation scattered along ducts that exhibited a K18+ER−PR−CD24highCD49flow profile and progenitor properties. In contrast, MMTV-Wnt1 induced canonical signaling in K14+ basal cells with CD24/CD49f profiles characteristic of two distinct stem/progenitor cell-types. MMTV-Wnt1 produced additional profound effects on multiple cell-types that correlated with focal activation of the Hedgehog pathway. We document that large melanocytic nevi are a hitherto unreported hallmark of early hyperplastic Wnt1 glands. These nevi formed along the primary mammary ducts and were associated with Hedgehog pathway activity within a subset of melanocytes and surrounding stroma. Hh pathway activity also occurred within tumor-associated stromal and K14+/p63+ subpopulations in a manner correlated with Wnt1 tumor onset. These data show MMTV-Wnt1 and MMTV-ΔN89β-catenin induce canonical signaling in distinct progenitors and that Hedgehog pathway activation is linked to melanocytic nevi and mammary tumor onset arising from excess Wnt1 ligand. They further suggest that Hedgehog pathway activation maybe a critical component and useful indicator of breast tumors arising from unopposed Wnt1 ligand.Canonical Wnt/β-catenin signaling regulates stem/progenitor cells and, when perturbed, induces many human cancers. A significant proportion of human breast cancer is associated with loss of secreted Wnt antagonists and mice expressing MMTV-Wnt1 and MMTV-ΔN89β-catenin develop mammary adenocarcinomas. Many studies have assumed these mouse models of breast cancer to be equivalent. Here we show that MMTV-Wnt1 and MMTV-ΔN89β-catenin transgenes induce tumors with different phenotypes. Using  Wnts are a family of secreted proteins that regulate tissue patterning and homeostasis. The canonical Wnt pathway operates by inhibiting proteolysis of cytoplasmic β-catenin, which enters the nucleus and regulates transcription through Lef/Tcf DNA binding partners. It is well documented that canonical Wnt/β-catenin signaling is required for the viability of particular stem cells, and forced activation of this pathway can expand stem/progenitors, alter cell fate and induce tumorigenesis Wnts are expressed throughout mammary development. Mice expressing Wnt inhibitors, or deficient in Lef-1, show defective embryonic mammary development, and loss of the Wnt coreceptor, LRP5/6, impairs postnatal development Wnt1 and stabilized β-catenin (ΔN89β-catenin) under the control of the mouse mammary tumor virus LTR (MMTV) induces precocious mammary development and adenocarcinoma formation Multiple lines of evidence demonstrate roles for Wnt/β-catenin signaling in mammary development and breast cancer −)/CD24low/CD29/49fhigh/ Sca-1−/keratin (K)14+ profile, that are enriched in “mammary repopulating units” (MRUs), which have a parent-progeny relationship with a second Lin−/CD24high/CD29/49flow subpopulation of alveolar-limited progenitors expressing a predominantly luminal K18+ profile high/CD133−/K18+ and Lin−/CD24+/CD29low/CD61+ K14+ profiles Recent studies have shown that only a minor subpopulation of human breast tumor cells can propagate tumors +/p63+) cells found exclusively within the Wnt-1 tumor microenvironment, as well as, in and around melanocytic hyperplastic nevi, which form a hallmark of all MMTV-Wnt1 mammary glands. These data show that Hh pathway activation is linked to Wnt-1-induced mammary tumor onset and nevi formation and suggest that Hedgehog pathway activation maybe a critical component and useful marker of breast tumors arising from unopposed Wnt1 ligand.Recently the connection between the physiological role of Wnt signaling in stem cells and its capacity to induce cancer when upregulated has been exploited to identify intestinal stem cells by virtue of their expression of a Wnt-responsive gene, Lgr5, that was initially found to be upregulated in colonic tumors + basal cells . In contrast, Wnt1 tumors were enriched for CD24lowCD49fhigh MECs (highCD49flow). To characterize cells undergoing an active transcriptional response to expression of these transgenes, we crossed MMTV-Wnt1 and MMTV-ΔN89β-catenin lines to the Axin2-d2EGFP reporter mouse, isolated tumor MECs from the bitransgenic progeny and analyzed them for enhanced green fluorescent protein (EGFP) and CD24/CD49f expression by flow cytometry. In contrast to the even distribution of EGFP− cells from MMTV-ΔN89β-catenin mice between the two major CD24/CD49f cell populations (hi (top 15%)  from tumors of MMTV-ΔN89β-catenin and MMTV-Wnt1 mice and investigated their expression of CD24 and CD49f. These markers separate MECs from D49fhigh . The moslow MECs    fell excpulation . Immunofhers K14 . We conct, EGFP−  and EGFPGFP− . Gli1 is both a transcriptional target gene and positive amplifier of the pathway. Its expression is strictly dependent upon Hh signaling and thus provides a reliable reporter of Hh pathway activation +/lzGli1 control glands and Gli1-lacZ reporter expression was restricted to lymphatics  and MMTV-ΔN89β-catenin (n = 5) mice and from all albino mice regardless of genotype . Mammary whole-mounts  (+/lzGli1 mice (n = 4) (+/lzGli1 mice (n = 4)  and control +/lzGli1 (n = 2) glands. We conclude that MMTV-Wnt1 expression induces aberrant melanocyte accumulation and differentiation within the fatpad and this event does not involve canonical pathways but correlates with robust Hh pathway activity within the stroma and a minor melanocyte subpopulation.To investigate whether Wnt1 induces these effects on melanocytes through canonical signaling and/or involves Hh pathway activation, we examined whole mounts of bitransgenic MMTV-Wnt1;s reside . In cont (n = 8) , colocal (n = 4)  and with (n = 4) . Gli1-laThe key findings of this study are that MMTV-ΔN89β-catenin and MMTV-Wnt1 induce distinct tumors, activate canonical signaling in disparate cell-types, and MMTV-Wnt1 alone results in focal Hh pathway activity within the mammary tumor microenvironment and in and around melanocytic hyperplasia.lacZ. These β-catenin-responsive cells display a hormone-receptor-negative +CD24highCD49flow profile, and generate colonies containing both K8+ and K14+ cells. Thus, they exhibit the expected location, molecular profile and functional bipotency ascribed to alveolar progenitor populations from normal mammary glands Previous studies have shown that MMTV-ΔN89β-catenin and MMTV-Wnt1 tumors express primitive cell markers and are enriched in cell populations with stem/progenitor features + cell population. Several pieces of data suggest that this Wnt1-responsive K14+ cell population consists of a stem cell or early bipotent progenitor population. First, the EGFPhi expressing cells within the CD24lowCD49high subpopulation is skewed towards higher CD49f expression, a feature associated with MRU capacity, and depleted of cells with lower CD49f expression, a feature associated with mature myoepithelial populations hi responding cells that segregate into the CD24highCD49low group are also skewed rightwards and express K14, a profile recently ascribed to a CD61+ bipotent progenitor population shown to have cancer stem cell properties Many studies have assumed that MMTV-ΔN89β-catenin and MMTV-Wnt1 exert a similar tumorigenic effect and have studied these models interchangeably. Where phenotypic differences have been noted Wnt1's potential to activate non-canonical pathways has been speculated upon but not experimentally addressed. Our results show that one definitive source of disparity between these models lies in the failure of Wnt1 to induce autocrine canonical signaling and its ability instead to induce a robust paracrine canonical response within a basally located K14wt or MMTV-ΔN89β-catenin mice. Although both canonical and non-canonical Wnt signaling have been implicated in promoting melanocyte proliferation, migration and differentiation at other body sites lacZ reporter expression within the mammary melanocytic hyperplasia strongly favors non-canonical signaling. Intriguingly, our results show a tight correlation between positive Hh signaling within a minor melanocytic subpopulation and their surrounding stromal neighbors and the massive accumulation of mobilized, differentiating melanocytes in MMTV-Wnt1 glands. Current literature highlights a dualistic role for Hh signaling in melanocyte biology A further source of disparity between the MMTV-ΔN89β-catenin and MMTV-Wnt1 phenotypes relates to the ability of the latter to affect additional surrounding cell-types. Of note, stromal hyperplasia see  and 3E iGli1-lacZ is expressed within a minor subset of p63+ basal cells in Wnt1 tumors and Ihh has been proposed to regulate proliferation of progenitors from stem cells via regulation of p63 isoform expression and to promote human mammosphere formation, considered to be a surrogate in vitro assay of stem cell proliferation A second major site of focal Hh pathway activity is found within MMTV-Wnt1 tumors. Of note, Hh pathway activity was restricted to the Wnt-1 tumor microenvironment and was not observed within the Wnt-1 hyperplasia and thus correlated with Wnt1 tumor onset. Hh pathway repression is essential for embryonic mammary development and repression remains critical to homeostasis in the postnatal gland In summary our data show that Wnt1 and ΔN89β-catenin generate distinct tumors at least in part through activating canonical signaling in distinct progenitors and through Wnt-specific paracrine effects on multiple cell-types. Our data also show that Wnt 1 expression is linked with focal Hedgehog pathway activation in melanocytic nevi and mammary tumors. These data suggest Hh pathway activity and pigmentary changes may have utility as indicators of breast tumor onset induced by excess or unopposed Wnt ligand.Animal maintenance and experimental procedures were in accordance with the NIH Guidelines for Animal Care and Use and were approved by the Institutional Animal Care and Use Committee of New York University Medical School.+/lzConductin, Gli1+/lz, +/lzLtpb1L were provided by Drs. Franke Costantini, Columbia University, Alexandra Joyner, Sloan Kettering Institute and Velocigene, Regeneron Pharmaceuticals, Inc. MMTV-Wnt1 and MMTV-ΔN89β-catenin mice were as described lacZ expression, mammary glands and tumors were fixed in 4% paraformaldehyde (PFA) in 1× phosphate buffered saline (PBS)  for 1 h, washed thrice in rinse buffer  for 1 h each and stained overnight at room temperature in X-gal staining solution  . After staining, glands and tumors were rinsed in 1× PBS, post-fixed in 4% PFA overnight and processed for paraffin embedding. The tissues were sectioned, counterstained with Nuclear Fast Red  and mounted for histological analyses. For whole mount analysis, X-gal stained mammary glands were counterstained with carmine, cleared and mounted as described http://mammary.nih.gov/tools/.To detect Tissues were either fixed in PFA and X-gal stained as described above or fixed in 10% phosphate buffered formalin overnight, then processed and embedded in paraffin. 4 µ sections were deparaffinized with xylene  and rehydrated through a graded series of ethanol. Citric acid antigen retrieval was performed for all antibodies by microwaving in 6.53 mM sodium citrate pH 6.0 for 30 min at 1.21 kilowatts. Rabbit antibodies against K14  (1∶2000), MUC1 (1∶500) (Abcam), laminin (1∶100) (Sigma), S-100β (1∶4000)(Dako #Z0311), mouse antibodies against K18 , estrogen receptor (1∶500) , and guinea-pig anti-vimentin (1∶1000)(Progen) were detected using biotin labeled secondary antibodies  (1∶1000) in conjunction with streptavidin peroxidase  that was colorimetrically detected using diaminobenzidine . For S-100β staining, sections were subjected to antigen retrieval with 0.1% trypsin (Sigma #T0303), 0.1% CaCl2 in 50 mM Tris-HCl, pH 7.6 at 37°C for 45 min in a humidified chamber. Slides were washed continuously under running tap water for 5 min and processed for immunohistochemistry. Azure B staining was performed as described −1 collagenase and 100 U ml−1 hyaluronidase. Digests were pipetted 8–12 times and vortexed for 5 seconds every hour. After dissociation, cells were pelleted at 350×g for 5 min and resuspended in a 1∶4 mixture of Hanks' Balanced Salt Solution containing 2% FBS (HF) and ammonium chloride and centrifuged again. Cell pellets were gently dissociated for 3 min in 5 ml of pre-warmed 0.25% trypsin-EDTA. 10 ml of cold HF was added. Cells were pelleted by centrifugation and resuspended for 1 min in 5 mg ml−1 of pre-warmed dispase II containing 0.1 mg ml−1 DNase I, then diluted with 10 ml of cold HF and filtered through a 40-µm mesh. All reagents were from StemCell Technologies Inc. 6 per 100 µl of 2% bovine serum albumin (BSA) were incubated on ice for 30 min with primary antibodies: 0.5 ng/ml of biotinylated TER119 (cat# 553672), CD31 (cat# 558737), CD45 (553078), CD24-phycoerythrin (PE) (cat# 553262), and 0.25 ng/ml of CD49f-allophycocyanin (APC) . Cells were washed once in 200 µl 2% BSA, stained with 1.5 ng/ml of streptavidin-Alexa488 on ice for 30 min, washed again and resuspended in 2% BSA. 1 µg/ml of 7-aminoactinomycin D (7-AAD)  was added just prior to data acquisition. Flow cytometric data was acquired on a FACS Caliber cytometer at the NYU Skirball FACS Core Facility and analyzed using FlowJo software Mammary glands and tumors were minced and digested for 6–7 h at 37°C in EpiCult-B Medium supplemented with 5% fetal bovine serum (FBS) containing 300 U ml4 cells/well in 24-well plates . Lineage+ cells were depleted prior to sorting using the EasySep Cell Biotin Selection Kit (StemCell Technologies Inc.) using TER119, CD31, and CD45 and CD140A (eBioscience) antibodies. GFP+, GFP− and Total Sorted tumor cells were sorted into 1 ml of 100% FBS using a MoFlo cell sorter, excluding cell debris, cell doublets, and autofluorescent cells. Sorted cell populations were centrifuged at 350 g for 3 mins, resuspended in a 300 µl, assessed for viability in 50% Trypan Blue and plated at limiting dilution. Media  was changed 48 h later to medium lacking FBS. Colonies were fixed after 6 days in 50∶50 acetone∶methanol at −20°C for 3 min and counted. Cells were stained with primary antibodies to K8 (Progen Cat#65138) and K14  for 30 min, washed three times in PBS, stained with goat anti-mouse  and donkey anti-rabbit  followed by three washes in PBS.NIH-3T3 cells were irradiated in suspension at 20Gy and seeded as a feeder layer 24 h prior to assay, at a concentration of 1.9×10


Therefore, possible targets for the anti-CRC activity of nonsteroidal anti-inflammatory drugs (NSAIDs) are β-catenin and catenin-related transcription (CRT). We tested the antiproliferative activity and the effects on levels of β-catenin and cyclin D1 protein, as well as CRT (measured using a synthetic β-catenin/TCF-reporter gene [TOPflash]), of a panel of NSAIDs  on SW480 human CRC cells in vitro. Following NSAID treatment, there was no consistent relationship between reduced cell proliferation, induction of apoptosis and changes in β-catenin protein levels or CRT. All the NSAIDs, except rofecoxib, decreased nuclear β-catenin content and cyclin D1 protein levels in parallel with their antiproliferative activity. However, cyclin D1 downregulation occurred prior to a decrease in total β-catenin protein levels and there was no correlation with changes in CRT, suggesting the existence of CRT-independent effects of NSAIDs on cyclin D1 expression. In summary, NSAIDs have differential effects on β-catenin protein and CRT, which are unlikely to fully explain their effects on cyclin D1 and their antiproliferative activity on human CRC cells in vitro.Elevated  Adenomatous Polyposis Coli (APC) is believed to occur prior to development of the benign precursor lesion of colorectal cancer (CRC), the colorectal adenoma (or polyp), in the majority of cases of sporadic colorectal carcinogenesis (β-catenin gene CTNNB1) leads to decreased β-catenin degradation and a subsequent increase in cytoplasmic and nuclear β-catenin protein levels  is a common early event in intestinal epithelial cells during human colorectal carcinogenesis . Loss ofogenesis . Loss ofn levels . This, iogenesis . Therefoβ-catenin protein levels in human CRC cells in vitro and in neoplastic epithelial cells of dimethyl-hydrazine-induced murine colonic tumours in vivo (β-catenin/TCF-reporter gene TOPflash) in human CRC cells. However, it remains unclear whether modulation of β-catenin expression and/or CRT is a property shared by other NSAIDs. In our initial study, downregulation of β-catenin protein was not evident in human CRC cells treated with aspirin or the experimental selective cyclooxygenase (COX)-2 inhibitor NS-398  indomethacin decreases β-catenin levels and/or CRT to the well-characterised antiproliferative activity of these drugs on human CRC cells in vitro. Therefore, we tested the effects of a diverse panel of NSAIDs, with differing selectivity for the two COX isoforms . SW480 human CRC cells were cultured on tissue culture plastic at 37°C, in the presence of 5% CO2. Cells were routinely subcultured using 0.25% (w v−1) trypsin/1 mM ethylenediaminetetraacetic acid (EDTA) solution (Invitrogen). Cells were counted using a haemocytometer and viability was assessed by exclusion of 0.4% (w v−1) Trypan blue . 1 × 105 viable cells were seeded per 35 mm well and were incubated for 96 h before NSAID treatment was commenced.SW480 human sporadic CRC cells  were grown in RPMI 1640 medium plus GlutaMAX-I™ supplemented with 10% (v/v) foetal bovine serum (FBS), 1000 U mlM stock solution of diclofenac was prepared in sterile distilled water. The other drugs were prepared as 100 mM stock solutions in dimethyl sulphoxide . Control cultures contained either DMSO or distilled water at an equivalent (v v−1) dilution to that used for the highest concentration of each NSAID.Indomethacin and diclofenac were obtained from Sigma. Sulindac sulphide and sulindac sulphone were obtained from ICN Biomedicals, Basingstoke, UK. For some experiments, sulindac sulphide was also obtained from Sigma. Rofecoxib powder was a kind gift from Merck Sharp & Dohme Ltd., Hoddesdon, Herts., UK. A 100 mβ-catenin antibody (6F9) and mouse monoclonal anti-human β-actin antibody (AC15) were obtained from Sigma. Mouse monoclonal anti-human cyclin D1 (A12) was obtained from Santa Cruz Biotechnology . Horseradish peroxidase-conjugated rabbit anti-mouse IgG was obtained from DakoCytomation .Mouse monoclonal anti-chicken g, prior to resuspension in a small volume of medium for counting. Adherent cells were then harvested by trypsinisation and counted as above. Data are represented as the mean and standard error of the mean (s.e.m.) cell count (n=3), for each time point and drug concentration.For each experiment, nonadherent and adherent cells in three individual wells were counted after a 96 h culture period (denoted time 0 h). Old medium was removed from the other wells and fresh medium containing a known concentration of NSAID or solvent carrier alone, was added to triplicate wells and incubated for 24, 48 or 72 h. At each time point, medium containing nonadherent cells was aspirated from triplicate wells and centrifuged for 5 min at 200 β-catenin was performed on adherent cells on coverslips as described  paraformaldehyde in phosphate-buffered saline (PBS). Fixed cells were resuspended in PBS containing 0.5 μg ml−1 Hoechst 33258 . The percentage number of morphologically apoptotic cells was counted by fluorescence microscopy as described . were placed in individual wells of a flat-bottomed 96-well plate and cultured for 96 h in order to mirror the experimental protocol used for cell counting assays. Cells were treated with NSAIDs or an equivalent dilution of carrier solvent for 48 h before addition of caspase substrate. Caspase-3/-7 activity was measured at 90 min by a fluorescence plate reader  as per the manufacturer's instructions . Data are presented as the mean (+s.e.m.) percentage fluorescence value for each NSAID compared with the appropriate carrier control (n=3). None of the NSAIDs had any direct activity on the caspase substrate in ‘no cells’ control wells (data not shown).SW480 human CRC cells  buffer (pH 7.2) containing 0.137 M sodium chloride , 1% Brij 96 , 1 mM EDTA and Complete™ protease inhibitor cocktail . Whole cell lysates were prepared as described , using 10% NuPAGE Bis-Tris 1.0 mm gels  as described (−1) dried skimmed milk powder in PBS (blocking solution) containing 0.05% (v v−1) Tween 20 (Sigma) for 1 h at 20°C. Membranes were probed with primary antibodies against β-catenin (1/5000 dilution) or β-actin (1/2500 dilution) in blocking solution for 1 h at 20°C. Anti-cyclin D1 antibody (1/500 in blocking solution) was applied overnight at 4°C. Horseradish peroxidase-conjugated secondary antibody (1/5000 or 1/1000 [for cyclin D1] dilution) in blocking solution was incubated with the membrane for 1 h at 20°C. Immunoreactive protein was detected using ECL chemiluminescence .Adherent cells were lysed in 50 mβ-catenin band to the β-actin band was determined. Each β-catenin/β-actin ratio was then expressed as a percentage of the control β-catenin/β-actin ratio . Data are represented as the mean (and s.e.m.) percentage of the control β-catenin/β-actin ratio value (n=3), for each time point and drug treatment.A Bio-Rad Molecular Imager FX was used to quantify individual bands. The background intensity was subtracted and the bands were quantified using Quantity One software (Bio-Rad). The relative intensity of the β-catenin/TCF-reporter construct TOPflash  with pRL-TK . The GeneJuice™-DNA mix was incubated for 30 min at 20°C before incubation with cells for 3 h at 37°C. The transfection medium was removed and fresh culture medium, containing drug or carrier control, was added. After incubation for 48 h at 37°C, cells were washed once with PBS, lysed with Passive Lysis Buffer and Dual-Luciferase reporter assays (both Promega) were performed as per the manufacturer's instructions. Firefly (TOPflash or FOPflash) luciferase activity was corrected for Renilla luciferase activity (pRL-TK) to control for transfection efficiency. In order to exclude any CRT-independent effect on thymidine kinase (tk) minimal promoter activity of TOPflash, TOPflash activity was normalised to the ratio of the FOPflash  activity between NSAID-treated and control cells, termed the FOPflash ratio. Data are expressed as the mean (and s.e.m.) of triplicate values of the normalised TOPflash activity, with the corresponding drug-treated/control cell FOPflash activity ratio.Experiments on the effect of NSAIDs on CRT, using the β-catenin/β-actin protein ratio and normalised TOPflash activity. The Bonferroni test was used for post hoc comparison of the effects of particular NSAID concentrations compared with control cell cultures. Statistical significance was assumed if the P-value was less than or equal to 0.05.One-way analysis of variance (ANOVA) was used to determine the significance of the effect of different concentrations of NSAIDs on cell number, the β-catenin protein levels, in four human CRC cell lines, regardless of the presence of mutant APC alleles or COX-2 expression , as well as decreased The viability of adherent SW480 human CRC cells was consistently greater than 95%. Therefore, the number of adherent cells at each time point was used to measure net cell proliferation. We, and others, have previously demonstrated that, following NSAID treatment of human CRC cells, the number of nonadherent cells accurately reflects the degree of apoptosis that has occurred , which did not increase during the 72 h experiment  did not induce caspase activation in SW480 human CRC cells at 48 h compared with control cells  was approximately half that of sulindac sulphone (200 μM). Treatment with sulindac sulphide at concentrations at and above 200 μM was associated with a pronounced increase in apoptosis  in caspase-3/-7 activity  had little effect on the proliferation of SW480 human CRC cells compared with the other NSAIDs that were tested in this study . However, a higher concentration of sulindac sulphide (300 μM) induced an apparent increase in nuclear β-catenin protein content  was associated with reduced nuclear β-catenin protein (data not shown) consistent with a previous report  concentrations of NSAIDs  concentrations of sulindac sulphone did not occur in association with significant changes in β-catenin protein content , prior to treatment with NSAIDs. TOPflash activity in control SW480 human CRC cells was consistently greater than 40-fold higher than the corresponding FOPflash activity, confirming significant basal CRT in cells lacking functional APC alleles. In order to exclude nonspecific effects of NSAIDs on gene expression, TOPflash activity in NSAID-treated SW480 human CRC cells was corrected for any drug-induced effects on FOPflash . NSAID treatment was associated with numerically small, variable effects on FOPflash activity, which are denoted as the ratio of FOPflash activity in treated vs control SW480 human CRC cells  of indomethacin (β-catenin protein levels (sulindac sulphone and rofecoxib) was not associated with reduced CRT  was associated with decreased caspase-3/-7 activity in SW480 human CRC cells. Further investigation of whether sulindac sulphide has direct caspase inhibitory activity is now required.Although caspase-3 cleavage has been linked to sulindac-induced apoptosis of human CRC cells β-catenin protein levels of varying degrees , as well as cyclin D1 mRNA and protein levels, in SW948 human CRC cells  of sulindac for 24 h was associated with increased ‘Tcf activity’ based on an increase in the TOPflash/FOPflash ratio (μM for 48 h) has been demonstrated to have no inhibitory effect on TOPflash activity in HEK293 cells (Our data on the effect of indomethacin on TOPflash activity conflict with those of β-catenin protein, cyclin D1 protein and CRT in human CRC cells. There is no simple relationship between alterations in total β-catenin protein levels and nuclear β-catenin localisation induced by NSAIDs and changes in CRT. Evidence is emerging that NSAIDs may also modulate WNT signalling via changes in the phosphorylation status of β-catenin and glycogen synthase kinase-3 (in vivo are now required in order to investigate further the activity of NSAIDs against this pivotal signal transduction mechanism in colorectal carcinogenesis.In summary, NSAIDs have differential effects on


Staphylococcus aureus (S. aureus), is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome. Ortho-phenylphenol (OPP) is an antimicrobial agent and an active ingredient of EPA-registered disinfectants with wide human exposure in various agricultural, hospital and veterinary disinfectant products. Despite many uses, an understanding of a cellular response to OPP and it's mechanism of action, targeted genes, and the connectivity between targeted genes and the rest of cell metabolism remains obscure.S. aureus when exposed to 0.82 mM of OPP for 20 and 60 min. Our data indicated that OPP downregulated the biosynthesis of many amino acids, which are required for protein synthesis. In particular, the genes encoding the enzymes of the diaminopimelate (DAP) pathway which results in lysine biosynthesis were significantly downregualted. Intriguingly, we revealed that the transcription of genes encoding ribosomal proteins was upregulated by OPP and at the same time, the genes encoding iron acquisition and transport were downregulated. The genes encoding virulence factors were upregulated and genes encoding phospholipids were downregulated upon 20 min exposure to OPP.Herein, we performed a genome-wide transcriptome analysis of the cellular responses of Staphylococcus: OPP inhibits anabolism of many amino acids and highly downregulates the genes that encode the enzymes involved in the DAP pathway. Lysine and DAP are essential for building up the peptidoglycan cell wall. It was concluded that the mode of action of OPP is similar to the mechanism of action of some antibiotics. The discovery of this phenomenon provides useful information that will benefit further antimicrobial research on S. aureus.By using microarray analysis that enables us to simultaneously and globally examine the complete transcriptome during cellular responses, we have revealed novel information regarding the mode of action of OPP on  S. aureus, which is responsible for many infectious diseases, ranging from benign skin infections to life-threatening endocarditis and toxic shock syndrome [The U.S. Environmental Protection Agency (EPA) has endeavored to determine the efficacy and the mode of action of antimicrobials. At EPA, 5,000 antimicrobial products are registered, and hospital-level disinfectants are being tested against pathogens such as syndrome . One of syndrome .The phenolic compound, ortho-phenylphenol (OPP), is an antimicrobial agent and an active ingredient of EPA-registered disinfectant with wide human exposure in various agricultural, hospital and veterinary disinfectant products. OPP is employed in a variety of applications, including hard surface disinfection, wood preservation, treatment of citrus fruit, vegetables before packaging to prevent microbial decay and textile production due to its bactericidal and fungicidal activity -5.There have been several reports related to the exposure of OPP on humans. It has been reported that OPP increased the incidence of urinary bladder tumors in F344/DuCrj rats when administered in the diet . The resMoreover, a lack of understanding of a cellular response to OPP hinders further development of more innovative methods for combating pathogens. Certainly, better elucidation of the molecular events responsible for establishing and maintaining pathogenicity will help to map affected cell functions and serve to delineate the mechanisms involved in the disinfectant activity.Pseudomonas aeruginosa and S. aureus upon exposure to antimicrobials [Microarrays have been effectively employed to simultaneously and globally examine the complete transcriptional response at the genomic level in crobials -15.S. aureus to OPP includes downregulation of genes involved in lysine metabolism, as well as genes involved in amino acid metabolism, by utilizing Affymetrix S. aureus GeneChip arrays. Our findings indicate that: (i) many cellular protective processes were upregulated, (ii) the transcription of genes involved in primary metabolic pathways was downregulated, and (iii) the transcription of genes encoding lysine and histidine biosynthesis was downregulated. Next we performed real-time PCR analysis on selected genes to validate the array results. Based on this result, it was concluded that this study may help to elucidate the mechanism of action by which OPP stops cell wall construction and thereby inhibiting S. aureus growth, and may facilitate the design of more effective antimicrobials.In this study, to our knowledge, for the first time, we show that the global transcription response of S. aureus, we first exposed the exponentially growing cells to different concentrations of OPP dissolved in DMSO (0 up to 1.18 mM). In figure S. aureus was reportedly 500 mg/l (3 mM) [S. aureus initially responds to OPP, we chose the rate of cell growth inhibition with 0.82 mM OPP after 20 and 60 min exposure times compared to control (without OPP).To determine the sublethal inhibitory effect of OPP on  0 up to .18 mM. Il (3 mM) . In thisp-value for a t-test should be equal to or less than 0.05, (ii) an absolute fold change in transcript level should be equal to or greater than 2, and (iii) a gene should have a presence or marginal call [S. aureus GeneChip, 2,348 genes showed statistical significance based on a 1-way ANOVA. We found that mRNA levels 669 genes of S. aureus were significantly altered in response to OPP by two fold or more upregulation or downregulation. The raw data of 7,775 genes control (0 min) and experimental genes after (20 and 60 min exposure to 0.82 mM of OPP) has been deposited in NCBI's Gene Expression Omnibus [To investigate early transcriptional changes in response to OPP exposure, we isolated total RNA after 20 min and 60 minutes exposure to 0.82 mM OPP and conducted five independent microarray experiments in the absence (control) and the presence  of 0.82 mM OPP   see als.S. aureus underwent the initial growth inhibition followed by partial growth recovery upon exposure to OPP.In Figure To further identify genes with similar transcription patterns during the time course, we removed 238 genes , hypothetical protein (132) and general function predicted only (70)). We categorized 431 genes with known functions into 6 groups on the basis of their transcription directions  gene expression upon both 20 and 60 min exposure. In our previous results, triclosan upregulated the expression of SA2423 encoding ClfB, which binds fibrinogen [S. aureus may be a secondary effect of OPP and this may provide new insight into the protective response of S. aureus to OPP.In this study, OPP also upregulated the brinogen . The resrplY) which encoded general stress protein was upregulated at 20 min. Ribosomal proteins are required for protein translation. Such early response of these ribosomal protein genes may reflect a stress response during exposure. The upregulation of ribosomal protein genes might enhance the translation process or help proper ribosome functioning under stress conditions as exposure to OPP. The suggestion that the expression of ribosomal proteins is activated upon exposure to OPP is surprising as this presumably reflects increased growth rate or virulence.Group II of table pyrRPBCAAABFE) which is homologous to the pyrimidine biosynthetic (pyr) operon of Bacillus subtilis [In group II table , we alsosubtilis  was upreS. aureus is an ABC transporter that transports amino acids, cations- and iron-carrying compounds and peptides with a broad specificity [S. aureus. The Opp system consists of four different proteins: OppB and OppC, OppD and OppF. Interestingly, 4 of the 26 genes, including SA0845–SA0848 that code for proteins associated with amino acid transport were upregulated (table potABCD) was upregulated , which are involved in the transport of spermidine and putrescine. Further, we showed the upregulation of ABC transport systems-related genes, which accompanied the growth recovery.In group III of table cificity . The peped table . Therefoed table . The potbacteria . The SA0crcB) is a putative integral membrane protein possibly involved in chromosome condensation , the heme-regulated transport system, which consist of a novel transport system which plays a critical role in staphylococcal heme metabolism (table lysC)-SA1226 (asd)-SA1227 (dapA)-SA1228 (dapB)-SA1229 (dapD), and SA1814 (dapE) fall within a predicted operon and are all involved in diaminopimelate (DAP) biosynthesis (table lysC)-SA1226 (asd)-SA1227 (dapA)-SA1228 (dapB) and SA1229 (dapD) show fold highest decreases as -54.6, -21.5, -27.3, -31.4, and -23.5 folds at 20 min and -7.7, -4.3, -5.2, -5.1, and -4.4 folds at 60 min in this experiment . This suggests that the mechanisms of action of 0.82 mM OPP on P. aeruginosa and S. aureus may differ.Intriguingly, we observed the high downregulation of SA2149 and SA2150 (dhoM)-SA1165 (thrC)-SA1166 (thrB) involved in threonine biosynthesis were also in this group (table ureABCEFD), which make CO2 and NH3 from urea and encode urea amidohydrolase and urease accessory proteins and SA1544 which codes for serine-pyruvate aminotransferase were downregulated at 20 and 60 min , SA1213 (opp-2C)-SA1214 (opp-2B), SA1659 (prsA), SA2235 (opuCC), and SA2409 which are cell wall anchoring surface proteins were downregulated in response to OPP (table Group IV of table bd complex: SA0937–SA0938 (cydAB) was downregulated upon both 20 min and 60 min exposure -SA1859 (ilvB) and SA1861 (ilvC)-SA1862 (leuA)-SA1863 (leuB)-SA1864 (leuC)-SA1865 (leuD)-SA1866 (ilvA) which were downregulated (table ilv-leu operon encoding enzymes of branched-chain amino acid biosynthesis in Bacillus subtilis [hisI)-SA2465 (hisF)-SA2466–SA2467 (hisH)-SA2468 (hisB)-SA2469 and SA2472 (hisZ) which are possibly parts of an operon homologous to the histidine biosynthesis were highly downregulated on 20 min (table trpD)-SA1202 (trpC)-SA1203 (trpF)-SA1204 (trpB)-AS1205 (trpA) which are also possibly parts of an operon homologous to the tryptophan biosynthesis operon are downregulated on 20 min (table S. aureus.In group V in table subtilis . In addimetQ) codes for probable D-methionine-binding lipoprotein (outer membrane lipoprotein 1). The proteins encoded by SA0344 (metE)-SA0769 (metN)-SA0770 (metI)-SA0420 (metN)-SA0421 (metI) are involved in D-methionine transporter of S. aureus ABC transporter -SA2476 (cbiO) are involved in cobalt and nickel transport -SA0145 (capB)-SA0146 (capC)-SA0147 (capD)-SA0148 (capE)-SA0149 (capF)-SA0150 (capG)-SA0151 (capH)-SA0152 (capI)-SA0154 (capK) were downregulated at 60 min. These genes share homology with the capsular polysaccharide synthesis enzyme (cap) operon which in turn is essential for virulence by impeding phagocytosis [capABCDEFGHIJ)) in S. aureus [S. aureus has demonstrated the importance of cell-wall sorted proteins in heme binding and transport [S. aureus. Cell-wall sorted proteins of the S. aureus iron-regulated surface determinant system bind human hemoproteins, remove the heme molecule, and transport heme through the cell wall and plasma membrane for accumulation in the bacterial cytoplasm [One of the characteristics of group VI of table ocytosis . This fi. aureus . Moreoveransport . To dateytoplasm .ribH)-SA1587 (ribA)-Sa1588 (ribB)-SA1589 (ribD) was downregulated at 60 min exposure  synthase, and β-subunit of riboflavin synthase [thiE)-SA1895 (thiM)-SA1896 (thiD) were involved in thiamine biosynthesis of coenzyme metabolism at 60 min -SA2150 (hrtB), which exhibited expression level decreases upon 20 and 60 min exposures in chorus with the repression of the genes of thiamine biosynthesis. Therefore, this result suggests that growth inhibition was accompanied with the repression of many coenzyme metabolism-related genes.Particularly important was that many of the genes in the class of "coenzyme metabolism" were also members of group VI figure . Intrigusynthase . These esynthase . The proica genes. Group VII is not indicated on figure icaADB) which make up the intercellular adhesion (ica) operon and contribute to virulence in S. aureus were downregulated after 20 min and upregulated after 60 min of exposure to OPP (table icaRADBC) mediates polysaccharide intercellular adhesion in S. aureus, which leads to cell-cell adhesion and is required for biofilm formation [S. epidermidis [Note that group VII has been included only in table ormation  Prior stdermidis ,46, and dermidis . TherefoS. aureus. Table As an independent measure of differential gene expression, we examined the relative levels of 18 genes with a range of fold changes (-265.0- to 24.5-fold) by real-time PCR analysis, which were specifically involved in the pathogenesis or metabolism of S. aureus, for the first time, by utilizing whole-genome microarrays. Moreover, we presented how the transcriptome profile of S. aureus was shifted during its cellular response to OPP, which involved the growth inhibition. To our knowledge, this is the first study demonstrating the activation of fermentative metabolism after OPP treatment in S. aureus. In summary, we revealed that amino acid metabolism genes were selectively downregulated between 20 and 60 minutes when exposed to 0.82 mM OPP. We also found that the growth inhibition was accompanied by the downregulation of many membrane function-related genes; however, the majority of these genes returned to normal transcription levels during the growth resumption. Further, we showed that the repression of the iron-regulated surface determinant (Isd) system, hemin and thiamine-related genes accompanied with the growth inhibition. Notably, we discovered the upregulation of virulence genes and ribosomal genes while the cells returned to normal growth. These results suggest that S. aureus might be arrested upon exposure to OPP.In this paper, we demonstrated how OPP upregulated and downregulated genes in S. aureus might be attributed to the inhibition of genes of lysine biosynthesis and subsequently peptidoglycan biosynthesis. We can therefore, conclude, that the mode of action of OPP is similar to the mechanism of action of some antibiotics. This study has revealed novel information on the mechanism of action of OPP in S. aureus which will benefit further antimicrobial research on S. aureus.In this study, OPP treatment led to the downregulation of several genes involved in amino acid anabolism. The genes involved in the DAP and lysine biosynthetic pathways were most significantly downregulated. Lysine and DAP are essential for building up the peptidoglycan cell wall. This finding proposes that the mode of action of the antimicrobial, OPP in S. aureus NCTC 8325 obtained from the Network on Antimicrobial Resistance in S. aureus (NARSA). As previously described [S. aureus cultures at 37°C with shaking at 250 rpm using sterilized Lurria-Bertani (LB) broth. For growth inhibition, 0.14 mg/L (0.82 mM) of OPP  was dissolved in DMSO and used for the microarray study and added immediately after OD600 reached 0.8. OD600 was measured by using Lambda 25 spectrophotometer . Note that the pH of S. aureus cultures was around 7.0 at 37°C after the exposure [In this study, we used escribed -12, we iexposure .Total RNA was isolated after 20 and 60 min incubation with and without (control) OPP using the RiboPure – Bacteria kit  . The quaS. aureus GeneChip arrays .cDNA synthesis, cDNA fragmentation, labeling, hybridization, staining and washing steps were performed according to the manufacturer's protocol for the Affymetrix alpha 1, 0.04; alpha 2, 0.06; tau, 0.015; target signal, 500. Fold changes were calculated as the ratio between the signal averages of five biological controls (untreated) and five biological experimental (OPP-treated) for 20 and 60 min exposures.The arrays were scanned with the Affymetrix GeneChip Scanner 3000. To analyze the array data, GeneChip Operating Software (GCOS) v. 1.2  and GeneSpring GX v. 7.3  were utilized with the following parameters: To determine the validity of the array data, transcript level changes obtained with the microarray analysis were compared with those from quantitative real-time PCR. Genes and primer sequences employed for the real-time PCR analysis are listed in table HJ performed microarray experiments, and data analysis, and drafted the manuscript. FT initiated and supervised the study, and reviewed the manuscript. CN and WEB reviewed the manuscript.The raw data of 7,775 genes control (0 min) and experimental (after 20 and 60 min. exposure of OPP). It has been also deposited in NCBI's Gene Expression Omnibus  and is accessible through GEO Series accession number GSE10605 .Click here for fileStaphylococcus aureus 669 genes that showed statistically significant mRNA level changes upon either 20 or 60 min exposure to OPP. The genes were grouped based on their regulation directions upon 20 and 60 min exposures.Click here for file


We have recently reported that mitogen-activated protein kinase (MAPK) JNK1 downregulates β-catenin signaling and plays a critical role in regulating intestinal homeostasis and in suppressing tumor formation. This study was designed to determine whether JNK2, another MAPK, has similar and/or different functions in the regulation of β-catenin signaling.in vitro system with manipulation of JNK2 and β-catenin expression and found that activated JNK2 increased GSK3β activity and inhibited β-catenin expression and transcriptional activity. However, JNK2-mediated downregulation of β-catenin was blocked by the proteasome inhibitor MG132 and GSK3β inhibitor lithium chloride. Moreover, targeted mutations at GSK3β phosphorylation sites (Ser33 and Ser37) of β-catenin abrogated JNK2-mediated suppression of β-catenin. In vivo studies further revealed that JNK2 deficiency led to upregulation of β-catenin and increase of GSK3-β phosphorylation in JNK2-/- mouse intestinal epithelial cells. Additionally, physical interaction and co-localization among JNK2, β-catenin and GSK3β were observed by immunoprecipitation, mammalian two-hybridization assay and confocal microscopy, respectively.We used an in vitro and in vivo, in which GSK3β plays a key role, although previous studies have shown distinct functions of JNK1 and JNK2. Our study also provides a novel insight into the crosstalk between Wnt/β-catenin and MAPK JNKs signaling.In general, our data suggested that JNK2, like JNK1, interacts with and suppresses β-catenin signaling  The Wnt/β-catenin signaling plays a critical role in embryonic development and the regulation of homeostatic self-renewal in a number of adult tissues Recently, an increasing body of evidence supported the crosstalk between MAPK and Wnt/β-catenin signaling JNK1, JNK2, and JNK3. JNK1 and JNK2 are ubiquitously expressed, whereas the expression of JNK3 is largely restricted to brain, heart, and testis The JNKs are encoded by three genes, namely Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco's modified Eagle's medium, human lung cancer cells A549 were grown in minimal essential medium. pcDNA3-Flag-MKK7-JNK2 (active JNK2) and pcDNA3-Flag-MKK7-JNK1 (active JNK1) were provided by Dr.Davis Standard immunoblotting analysis was used as described Renilla luciferase activity assay using a dual luciferase kit (Promega). The TCF-4 reporter activity was presented as by ratio of firefly to Renilla luciferase activity. Each experiment was triplicated independently.Cells were co-transfected with pcDNA3-HA-β-catenin and pcDNA3-Flag-MKK7-JNK1 or pcDNA3-Flag-MKK7-JNK2, along with TOPFLASH or FOPFLASH. 24 or 48 h after transfection, the cells were harvested to conduct a firefly and Similar as we described previously As described 5 cells/well in 24-well plate the day before transfection. Two µg of pACT-JNK2, pBind-β-catenin and pG5luc were co-transfected using Lipofectamine 2000 . pACT and pBind were used as negative controls. To eliminate non-specific interactions, two groups of negative controls were set: pACT-JNK2 and pBind, pACT and pBind-β-catenin. After 48 h, samples were lysed using 1x Passive lysis buffer, and the amount of firefly luciferase and Renilla luciferase was quantified using the Dual-Luciferase Reporter Assay System , as we described recently HEK293T cells were plated at a density of 2.5×10As described recently The studies from us and others have demonstrated that JNK1 can antagonize the canonical Wnt/β-catenin signaling To determine whether activated JNK2 can inhibit the aberrantly accumulated nuclear β-catenin in cancer cells, a human lung cancer cell line A549 was transfected with MKK7-JNK2. Immunoblotting analysis showed a reduction of endogenous β-catenin protein , which iβ-catenin interacts with TCF-4 and stimulates gene expression. To determine whether JNK2 activation can affect the β-catenin-driven gene transription, MKK7-JNK2 was co-transfected with β-catenin into HEK293T cells along with TCF-4 reporter plasmid TOPFLASH or the inactive control FOPFLASH. TOPFLASH includes a luciferase reporter driven by three copies of TCF binding elements upstream of the thymidine kinase (TK) minimal promoter, and is specifically regulated by Wnt/β-catenin signaling To confirm the suppression of Wnt/β-catenin signaling by JNK2 activation, a constant level of β-catenin was co-transfected with increasing amounts of MKK7-JNK2 into HEK293T cells. The alterations of exogenous β-catenin were assessed by immunoblotting. As shown in Cytoplasmic β-catenin protein levels are regulated by ubiquitination and subsequent proteasomal degradation. To determine whether the decrease of β-catenin protein induced by JNK2 activation is due to increased proteasomal degradation, a proteasomal inhibitor MG132 Multiple pathways have been reported to mediate β-catenin degradation by the proteasome, such as p53/Siah-1/APC, Wnt/GSK3β/APC, and retinoid X receptor (RXR)-mediated pathway To further test our hypothesis, GSK3β inhibitor lithium chloride (LiCl) was used to suppress GSK3β activity and the effect of activated JNK2 on β-catenin expression was analyzed. As shown in Post-translational modifications play critical role in the regulation of the β-catenin turnover by GSK3β Besides the indirect phosphorylation and degradation of β-catenin by JNK2 via GSK3β from our present study, a recent study has identified three more phosphorylation sites in β-catenin protein, two of them (Ser-191 and Ser-605) are critical for β-catenin stabilization and nuclear localization, and could be directly phosphorylated by JNK2, providing more evidences of the different roles of JNK2 in Wnt/β-catenin signaling in vivo, JNK2-/- and +/+ mice were dissected. As shown in in vivo data provided additional evidence that GSK3β is involved in JNK2-mediated β-catenin regulation.To validate the regulation β-catenin by JNK2 in vitro study demonstrated that selenium-stimulated phosphorylation of JNK1/2 dramatically inhibited β-catenin and its downstream targets, such as c-myc, cyclin D1 and CDK4, in human colon cancer cells , supporting the notion of activated JNK1 and JNK2 in the suppression of β-catenin signaling.As to the effect of JNK2 on β-catenin downstream targets, our most recent Although our data strongly demonstrate that JNK2 suppresses β-catenin signaling through GSK3β-proteasome pathway, the mechanism underlying the regulation of GSK3β activity by active JNK2 remains elusive. In addition, we can not exclude the possibility that JNK2 can directly interact with β-catenin. Therefore, immunoprecipitation assays were carried out to test a potential interaction between JNK2, GSK3β, and/or β-catenin. As shown in Since overexpressed β-catenin is mainly restricted in the nucleus as shown in in vitro, and that JNK2 deficiency upregulated β-catenin signaling in vivo, in which GSK3β is likely to play a critical role. Our study also provides a novel insight into the crosstalk between Wnt/β-catenin and MAPK JNKs signaling.In conclusion, our present data demonstrate that activated JNK2 promoted β-catenin degradation and inhibited the canonical Wnt/β-catenin signaling


PTHrP expression. In addition, Wnt/β-catenin signaling regulates chondrocyte hypertrophy in a non-cell autonomous manner and Gdf5/Bmp signaling may be one of the downstream pathways. Furthermore, Wnt/β-catenin signaling also controls final maturation of hypertrophic chondrocytes, but such regulation is PTHrP signaling-independent.Sequential proliferation, hypertrophy and maturation of chondrocytes are required for proper endochondral bone development and tightly regulated by cell signaling. The canonical Wnt signaling pathway acts through β-catenin to promote chondrocyte hypertrophy whereas PTHrP signaling inhibits it by holding chondrocytes in proliferating states. Here we show by genetic approaches that chondrocyte hypertrophy and final maturation are two distinct developmental processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by inhibiting PTHrP signaling activity, but it does not regulate  Sox9 and Col2a1. Closer to the middle of the cartilage, proliferating chondrocytes become elongated and line up in columns along the longitudinal axis. These columnar chondrocytes divide quickly and begin to express Indian hedgehog (Ihh) and Runx2 as they exit the cell cycle to become prehypertrophic chondrocytes. Prehypertrophic chondrocytes then differentiate into hypertrophic chondrocytes that no longer express Sox9 and Col2a1, but express Col10a1 and angiogenic factors such as vascular endothelial growth factor (Vegf). Hypertrophic chondrocyes can be further divided into two populations according to their differences in gene expression and mineralization levels. Ihh and parathyroid hormone receptor 1 (Pthr1) are expressed only in prehypertrophic and early hypertrophic chondrocytes. The mature population of hypertrophic chondrocytes express Osteopontin (Opn) Mmp13) that is required for invasion of blood vessels and osteoblasts to form the trabecular bone Mammalian skeletal development is controlled by two mechanisms, endochondral and intramembranous bone formation. Endochondral ossification occurs in most parts of the body and requires a cartilage model prior to bone formation. Cartilage is composed of chondrocytes and in the developing long bones, these chondrocytes are organized into zones with distinct cellular morphologies and proliferation properties. Such organization is essential for the proper directional growth and elongation and is subject to tight regulation by secreted signaling molecules and transcription factors Ihh activates PTHrP expression and PTHrP then signals to proliferating chondrocytes to inhibit Ihh expression and chondrocyte hypertrophy by holding chondrocytes in a proliferating state. Wnt signaling pathways also regulate chondrocyte hypertrophy Ihh, parathyroid hormone related peptide (PTHrP), Wnts, Bone morphogenetic proteins (Bmps) and fibroblast growth factor (Fgfs) etc., regulate progressive chondrocyte proliferation and hypertrophy. PTHrP and Ihh are major regulators of chondrocyte hypertrophy by forming a negative feedback loop Here we have investigated the regulation of chondrocyte hypertrophy and final maturation and the genetic relationship between the canonical Wnt and PTHrP signaling pathways in sequential chondrocyte differentiation. We uncovered that chondrocyte hypertrophy and final maturation are two distinct processes that are differentially regulated by Wnt/β-catenin and PTHrP signaling. Canonical Wnt signaling promotes chondrocyte hypertrophy by antagonizing PTHrP signaling activity. However, the final maturation of hypertrophy chondrocytes is controlled by Wnt/β-catenin signaling independently of PTHrP signaling.PTHrP expression was regulated by Wnt/β-catenin signaling in the developing long bone cartilage. β-catenin was removed in chondrocytes using the Col2a1-Cre mouse line c/c;Col2a1-CreCatnb mouse embryos PTHrP expression in the β-catenin deficient cartilage was similar to that in the control, but weaker at 14.5dpc  versus Alcian blue stained segment  was compared in the same cartilage elements , which encodes the signaling receptor for Ihh, in the cartilage in the c/c; Col2a1-CreSmo embryo PTHrP expression was diminished in the cartilage, but still detectable in the joint region in the c/c; Col2a1-CreSmo embryo  Cre line, Col2a1-CreEREx3/+; Col2a1-CreERCatnb embryos following TM injection. Since Lef1 is a transcription target of Wnt/β-catenin signaling Lef1 expression was upregulated  and Bmp2 expression are upregulated by Wnt/β-catenin signaling As PTHrP signaling controls chondrocyte hypertrophy cell autonomously egulated . In addistaining , suggest-catenin . Howeverdrocytes . This isWe report here that during mouse embryonic cartilage development, Wnt/β-catenin signaling controls chondrocyte hypertrophy and final maturation by two distinct mechanisms. Wnt/β-catenin signaling regulates initiation of chondrocyte hypertrophy by antagonizing PTHrP signaling, whereas it acts independently of PTHrP signaling in controlling the final maturation of hypertrophic chondrocytes . Our resc/c; Col2a1-CreCatnb mutant embryo is due to upregulated PTHrP signaling, not enhanced PTHrP expression. As β-catenin is required for activating the expression of downstream targets of the canonical Wnt signaling pathway, a possible scenario is that one of the transcription targets of the canonical Wnt signaling pathway in the chondrocytes suppresses the PTHrP signaling activity. Alternatively, the β-catenin protein may directly suppress PTHrP signaling activity by interacting with PTHrP signaling components. Interaction of β-catenin with components of other signaling pathways may be an important mechanism underlying Wnt signaling cross regulation with other pathways. For instance, a LEF/TCF independent regulatory role of β-catenin in cell lineage determination has been demonstrated during pituitary gland development, in which β-catenin binds a specific homeodomain factor, Prop1, to activate expression of the critical lineage-determining transcription factor, Pit1 Col2a1 nor Col10a1. Interestingly, PTH activation of Pthr1 has been shown to activate the Wnt/β-catenin signaling directly by stabilizing β-catenin protein in osteoblasts Our data suggests that delayed chondrocyte hypertrophy in the Wnt/β-catenin signaling has two distinct roles in cartilage development. First, it inhibits chondrocyte cell lineage determination and maintenance PTHrP null allele +/−PTHrPβ-catenin conditional allele c/cCatnb or gain-of-function allele ex3Catnb, and a transgene Cre which is under the regulation of Col2a1 promoter and enhancer. Embryos were collected at 14.5, 15.5 and 16.5 dpc and embryos were analyzed as described. Genotyping for the Col2a1-Cre transgene and β-catenin alleles were performed as previously described Embryos were generated by crosses between mice carrying heterozygous Alizarin red and Alcian blue staining of skeletons was conducted as previously described 35S-lablled riboprobes of Col 2a1, Col 10a1, Ihh, Pthr1, PTHrP, Opn, Mmp13, Mmp9 and Vegf were synthesized as previously Limbs were fixed by 4% paraformaldehyde at 4°C overnight, embedded in paraffin and sectioned at 6 µm. Sections were stained by Safranin O and Von Kossa method. In situ hybridization was performed as previously described 60 mg/ml Tamoxifen (Sigma) was dissolved in corn oil (Sigma) and sonicated until the solution became clear. The solution was filtered and 0.05 ml was injected intraperitoneally into the pregnant female mice at 13.5 dpc and the embryos were harvested at 17.5 dpc.Figure S1Analysis of chondrocyte hypertrophy in vertebral skeletons. Vertebral skeletons were visualized by Alcian Blue / Alizarin Red staining at E14.5. Mineralization was greatly reduced in the ribs and vertebral bodies in both the Catnbc/c; Col2a1-Cre single mutant and the PTHrP−/−; Catnbc/c; Col2a1-Cre double mutant. Chondrocyte hypertrophy was revealed by Safranin O staining and the expression of Col2a1 and Col10a1 in sections of vertebral bodies at E14.5. Compared to the Catnbc/c; Col2a1-Cre single mutant, chondrocyte hypertrophy in the PTHrP−/−; Catnbc/c; Col2a1-Cre double mutant was much accelerated and similar to that in PTHrP−/− single mutant embryos indicated by the expression of Col10a1.(3.47 MB TIF)Click here for additional data file.Figure S2Analysis of chondrocyte proliferation in PTHrP and β-catenin mutant embryos. (A) BrdU-labeled chondrocytes were detected by immunohistochemistry on sections of distal tibia at 16.5dpc. Zone I cells are resting chondrocytes and Zone II are columnary proliferating chondrocytes. Zone I and II were greatly reduced and the difference between them was not clear in the PTHrP−/− single mutant and PTHrP−/−; Catnbc/c; Col2a1-Cre double mutant embryos. The entire proliferating region was marked as Zone I and II. (B) The percentage of BrdU labeled chondrocytes was counted from four different samples of each genotype and the average with standard deviations are shown. Significant difference with p<0.05 is shown.(2.47 MB TIF)Click here for additional data file.Figure S3Analysis of osteoblast differentiation in PTHrP and β-catenin mutant embryos. Consecutive sections of developing humerus at E14.5 and E16.5 were examined by in situ hybridization with indicated probes. (A) At E14.5, expression of early osteoblast marker Runx2 and Osx in PTHrP−/−; Catnbc/c; Col2a1-Cre double mutant embryos was accelerated to the same level as that in PTHrP−/− mutant embryos. (B) At E16.5, expression of Runx2 and Osx in double mutant embryos was accelerated whereas Osc expression was still delayed compared to that in PTHrP−/− single mutant embryos.(4.01 MB TIF)Click here for additional data file.Figure S4Analysis of RANKL expression in PTHrP and β-catenin mutant embryos. Proximal tibia sections at E16.5 were examined by immunohistochemistry with a RANKL monoclonal antibody. The expression of RANKL by osteoblasts is indicated by an arrow. RANKL expression in mature hypertrophic chondrocytes was very low and slightly increased in the Catnbc/c; Col2a1-Cre mutant but not in the PTHrP−/− and PTHrP−/−;Catnbc/c;Col2a1-Cre mutant embryos.(3.75 MB TIF)Click here for additional data file.