The APC tumour suppressor functions in several cellular processes including the regulation of β-catenin in Wnt signalling and in cell adhesion and migration.In this study, we establish that in epithelial cells N-terminally phosphorylated β-catenin specifically localises to several subcellular sites including cell-cell contacts and the ends of cell protrusions. N-terminally phosphorylated β-catenin associates with E-cadherin at adherens junctions and with APC in cell protrusions. We isolated APC-rich protrusions from stimulated cells and detected β-catenin, GSK3β and CK1α, but not axin. The APC/phospho-β-catenin complex in cell protrusions appears to be distinct from the APC/axin/β-catenin destruction complex. GSK3β phosphorylates the APC-associated population of β-catenin, but not the cell junction population. β-catenin associated with APC is rapidly phosphorylated and dephosphorylated. HGF and wound-induced cell migration promote the localised accumulation of APC and phosphorylated β-catenin at the leading edge of migrating cells. APC siRNA and analysis of colon cancer cell lines show that functional APC is required for localised phospho-β-catenin accumulation in cell protrusions.We conclude that N-terminal phosphorylation of β-catenin does not necessarily lead to its degradation but instead marks distinct functions, such as cell migration and/or adhesion processes. Localised regulation of APC-phospho-β-catenin complexes may contribute to the tumour suppressor activity of APC. Adenomatous Polyposis Coli (APC) gene is responsible for over 80% of colon tumours, and is the first detectable event in colorectal neoplasia Mutation of the Evidence is accumulating that the mechanism of regulation of different cellular processes by β-catenin is more complex than simply involving the control of total cellular levels of the protein While several studies using phospho (Ser33/37/41/45)-specific β-catenin antibodies reinforce the concept that N-terminal phosphorylation of β-catenin leads to its degradation APC mutations 2+-dependent cell-cell contacts and with APC in clusters at microtubule ends in cell protrusions. Here, β-catenin associated with APC exists in a complex that is separate from axin and is rapidly phosphorylated and dephosphorylated by a GSK3β-dependent mechanism. Localised phospho-β-catenin/APC clusters accumulate in migrating cells and are lost in colon cancer cells when APC is truncated. The subpopulation of phospho-β-catenin at the microtubule tips appears to play a role in the tumour suppressor function of APC.In addition to regulation of β-catenin degradation, APC has been implicated in a range of cellular processes based on interactions with cytoskeletal proteins To investigate β-catenin phosphorylation, we examined the localisation of β-catenin in Madin-Darby Canine Kidney 3 (MDCK) epithelial cells using two phospho-specific β-catenin antibodies. One antibody was raised against β-catenin phosphorylated on Thr41 and Ser45 [P41/45] and the other was raised against phosphorylation at Ser33, Ser37 and Thr41 [P33/37/41]. We first confirmed that β-catenin is predominantly localised to cell-cell contacts in MDCK epithelial monolayers using a pan β-catenin antibody that recognises all molecular forms of β-catenin Fig S1). Immunofluorescent staining by P41/45- and P33/37/41-β-catenin antibodies at cell-cell contacts and in clusters was markedly reduced in β-catenin depleted cells , indicating that these populations represent phospho-β-catenin, although the nuclear signal remained, suggesting that it is not specific for phospho-β-catenin. Secondly, competition with phospho-peptides of the same sequence as the antigen used to raise phospho-β-catenin antibodies, competed for detection of distinct subcellular populations by P41/45- and P33/37/41-β-catenin, with only the nuclear signal remaining, again suggesting that the nuclear staining may be non-specific . Thirdly, phosphorylation of recombinant β-catenin in vitro demonstrates that P41/45 antibodies recognise β-catenin phosphorylated by CK1α alone or CK1α plus GSK3β; P33/37/41 antibodies recognise β-catenin phosphorylated by CK1α plus GSK3β but not by CK1α nor GSK3β alone . This data confirms that CK1α phosphorylates Ser45 while GSK3β is required for phosphorylation of Thr41, Ser37, Ser33. In addition, phosphorylation of Thr41 by GSK3β is not necessary for recognition by the P41/45 antibody, suggesting that it predominantly recognises phospho-Ser45. Neither antibody recognises unphosphorylated β-catenin . Finally, neither P41/45- nor P33/37/41-β-catenin antibodies detect β-catenin in interphase LIM2551 colon cancer cells that harbour a mutation resulting in deletion of the epitope for the phospho-β-catenin antibodies . We note that the P33/37/41 phospho-β-catenin antibodies also stain centrosomes and mid-body bridges in mitotic LIM2551 cells , indicating that the apparent localisation of phospho-β-catenin at centrosomes and concentrated at the mid-body during mitosis To test the specificity of P41/45- and P33/37/41-phospho-β-catenin antibodies for the different subpopulations, four control experiments were employed. Firstly, β-catenin was depleted by siRNA . Cells containing mutations in APC (LIM2537 and SW480) have detectable phospho-β-catenin whereas cells containing β-catenin mutations at Ser45 (LIM1899 and HCT116) do not contain phospho-β-catenin recognised by P41/45. However, the P41/45 phospho-β-catenin antibody still recognises T41A β-catenin (LIM1215) further demonstrating that the P41/45 antibody primarily recognises phospho-Ser45 rather than phospho-Thr41. Interestingly, Ser45 mutant β-catenin, which lacks the ability to be ‘primed’ for GSK3β phosphorylation by CK1α, is still recognised by P33/37/41 , consistent with previous findings Fig. S5). These data suggest that stable populations of N-terminally phosphorylated β-catenin are associated with different subcellular sites, and that, in contrast to prevailing models, β-catenin phosphorylation may not only be involved in degradation.We examined MDCK and 6 colon cancer cell lines and found that the major band detected by P41/45- and P33/37/41-phospho-β-catenin antibodies co-migrates with β-catenin (We then examined which proteins are associated with populations of phospho-β-catenin in different subcellular locations. β-catenin phosphorylated at Thr41 and Ser45 (as detected by the P41/45 antibody) is localised to cell-cell contacts with E-cadherin Fig. 2A.Fig. S6), in contrast to Maher et al We investigated the nature of peripheral phospho-β-catenin further by testing the impact of dissociation of calcium dependent adhesions. Chelation of extracellular calcium disrupts cell-cell contacts . The clusters of P33/37/41-β-catenin staining in Co-staining with APC reveals that P41/45- and P33/37/41-β-catenin cluster with APC at the ends of microtubules . P41/45 β-catenin is not affected by GSK3β inhibitors, as expected . The localisation of P41/45 β-catenin with APC clusters implies that phosphorylation at Ser33, Ser37 and Thr41 by GSK3β does not result in recruitment of β-catenin to APC clusters, but instead that β-catenin resident in these complexes is phosphorylated by GSK3β in situ. Surprisingly, the P33/37/41 β-catenin at cell contacts is not affected by inhibition of GSK3β even after long treatments . The loss of P33/37/41- and P41/45-β-catenin cell contact staining upon CK1 but not GSK3β inhibition  indicates that CK1, but not GSK3β, is involved in β-catenin phosphorylation at cell-cell adhesions. This implies that either CK1 phosphorylates P33/37/41 localised at the cell periphery or that S45 phosphorylation is required for these sites to be phosphorylated by another priming-dependent kinase. Thus, β-catenin is phosphorylated both by CK1 and GSK3β, but is selectively phosphorylated by different kinases at different sub-cellular sites.To test whether the different subpopulations of N-terminally phosphorylated β-catenin are GSK3β-dependent, we treated cells with two different GSK3β inhibitors. Both kenpaullone and LiCl treatment results in loss of APC-coincident P33/37/41 β-catenin clusters , which suggests that the loss of phospho-β-catenin upon GSK3β-inhibition is not due to protein turnover/degradation. P33/37/41-β-catenin coincident with APC at the ends of microtubules is reduced within 5 min of LiCl treatment and is depleted for several hours . P33/37/41-β-catenin co-incident with APC is also increased ). The rapid loss of phosphorylation upon treatment with LiCl indicates that a phosphatase may directly regulate β-catenin, as suggested in earlier studies Fig. S10B), there is a robust increase in the proportion of P33/37/41-β-catenin co-incident with APC ). Interpretation of these experiments is complicated by any direct effects of phosphatase inhibitors and LiCl treatment on the auto-inhibitory Ser9 phosphorylation of GSK3β as has been reported To investigate phosphorylation of β-catenin by GSK3β further, we first inhibited GSK3β by pretreatment with LiCl and then washed out the inhibitor and assessed P33/37/41-β-catenin levels. Consistent with rapid phosphorylation, P33/37/41-β-catenin increases after removal of LiCl from the media . Note that these accumulations of P33/37/41 are not coincident with APC.We then tested whether loss of APC impacts phospho-β-catenin localised to the ends of microtubules by depleting APC with siRNA. Transfection of MDCK cells with siRNAs targeting APC reduces the level of APC Fig. 7A To test the significance of APC in phospho-β-catenin localisation at cell extensions, we examined P33/37/41-β-catenin in colon cancer cells containing either intact or mutated APC. LIM2405 cells contain a single intact APC allele and harbour a mutation in the other allele which results in truncation at residue 2198 β-catenin is a critical component of cell-cell adhesions, where it forms a dynamic link between E-cadherin and the actin cytoskeleton 2+-dependent cell adhesion may reflect the increased transcriptionally active β-catenin reported by Maher et al 2+-dependent manner, it does not associate with E-cadherin by immunoprecipiation analysis, supporting the conclusion from Maher et al. N-terminally phosphorylated β-catenin localised to sites of cell-cell contact is associated with E-cadherin and requires intact cell-cell adhesions. Phospho-β-catenin has recently been reported in two separate complexes: one with E-cadherin and the other with components of the destruction complex The striking cytoplasmic distribution of phospho-β-catenin in mitotic cells, particularly for P41/45-β-catenin, suggests a role in cell-cycle regulation. Total β-catenin levels are reported to increase in early S phase and are maximal at G2/M 2+-dependent cell contacts and forms a complex with E-cadherin whereas phospho-β-catenin was not detected in VE-cadherin complexes Our discovery that phospho-β-catenin accumulates with APC clusters at the ends of microtubules reveals an unexpected role for β-catenin in conjunction with microtubule-associated APC. APC localisation to the plus ends of growing microtubules is well established We propose that the form of N-terminally phosphorylated β-catenin which accumulates with APC at cell extensions is not targeted for degradation but may regulate microtubule-associated APC function. Stabilised β-catenin has previously been implicated in inhibition of APC function in neurite formation and growth Our experiments show that GSK3β phosphorylates the β-catenin that accumulates with APC at microtubule ends. The action of GSK3β is intriguing. GSK3β phosphorylation of APC is reported to abolish microtubule binding The rapid loss of phospho-β-catenin at the protrusion tips following GSK3β inhibition suggests the presence of a phosphatase, as proposed in an earlier study While our data showing that axin is excluded from APC-rich protrusions suggests that phospho-β-catenin is not always targeted for proteasomal-mediated destruction, we cannot rule out a model whereby phosphorylation of β-catenin earmarks the protein for destruction by non-axin-mediated mechanisms. One such scaffold complex, containing presenilin, has previously been implicated in mediating phosphorylation of β-catenin Overall, our studies implicate phosphorylated β-catenin in cell adhesion and at the ends of APC-associated HGF- and wound healing-induced cell protrusions. The latter process requires functional APC and is perturbed in cancer cells containing mutated APC . RegulatThe anti-phospho-β-catenin P41/45 and P33/37/41 antibodies were from Cell Signaling . The APC monoclonal antibody (APC-NT) was raised against amino acids 1–61 of human APC with an N-terminal Flag epitope tag, produced in bacteria . Commercial antibodies were obtained from Santa Cruz (anti-APC (H290) and CK1α), Zymed (axin-1), BD Transduction Laboratories (β-catenin (19920/610153), GSK3β (G22320), E-cadherin, nucleoporin, PP2A), and Sigma . AlexaFluor-conjugated secondary antibodies were from Molecular Probes and IR Dye-conjugated secondary antibodies were from LiCor Biosciences.MDCK cells 2+ chelation experiments, MDCK cells were serum starved for 24 h and incubated with 4 mM EGTA, 1 mM MgCl2 in DMEM or control DMEM for 2 h. For GSK3β inhibitor experiments, cells were incubated with 2 nM Kenpaullone  or dimethyl sulfoxide (DMSO) control, 30 mM LiCl or 30 mM NaCl, for the indicated times. For inhibition of CK1, cells were incubated with 100 µM D4476  diluted as described For Ca5′-GGAAUCAACCCUCAAAAGUtt-3′, siAPC 2: 5′-GCACACUGCACUGAGAAUAtt-3′, siAPC 3 5′-GCACACUGCACUGAGAAUAtt-3′ (Ambion).To deplete APC using RNA interference (RNAi), MDCK cells were transfected with small interfering RNA (siRNA) directed against APC with Lipofectamine 2000  according to manufacturer's instructions using 0.3 µM siRNA targeting canine APC (Ambion) or 0.3 µM non-targeting siRNA. Predesigned Dharmacon siRNA pools targeting β-catenin  and non-target control siRNA pools (D-001810-20) were used to deplete β-catenin in MDCK cells. Cells were harvested for immunoblot analysis or fixed for immunofluorescence 48 h after siRNA treatment. The sequences of siRNA oligonucleotides were: siAPC 1: Cells were immunostained as described previously g for 30 min at 4°C. For immunoprecipitations, the lysates were incubated with the indicated antibodies (1–2 µg) for 16 h at 4°C and with protein A- or G-Sepharose (10% slurry) (Pharmacia) for the final 30 min 4°C. Precipitated proteins were eluted with 2× SDS-PAGE sample buffer and boiled for 10 minutes. Cell lysates and immunoprecipitated proteins were analysed by SDS-PAGE using 3–8% TrisAcetate or 4–12% Bis-Tris NuPAGE gels (Novex). Proteins were detected using the indicated primary antibodies and the Odyssey infrared imaging system (Odyssey). Protein bands were quantitated by densitometry, and analysed using GeneTools software.MDCK cells were lysed in ice-cold Lysis Buffer , and Complete (Roche) EDTA-free protease inhibitor cocktail). Lysates were clarified by microcentrifugation at 16,000 5 or 1×106) were placed in the upper compartment of a chamber  containing a 3.0-µm porous polycarbonate membrane insert. Cells were allowed to attach for 2 h with 2 ng/ml HGF (R and D Systems), and then inserts placed in chambers containing serum free DMEM or DMEM+10%FCS. Cells were allowed to extend pseudopodia through the pores for 24 h. Pseudopodia (PS) and cell bodies (CB) on the lower- and upper-surface of the filter were immunostained using standard methods, after first removing upper and lower-surface proteins, respectively, as described Pseudopodia were extracted as described by 5 cells were induced to extend protrusions across the filter. Proteins on the lower-(PS) and upper-(CB) surface of the filter were scraped directly into Lysis Buffer after first removing upper- and lower-surface proteins, respectively, by wiping the surface as described To extract proteins, 5×10Figure S1Reduction of phospho-β-catenin following β-catenin siRNA treatment MDCK cells were treated with siRNA directed against β-catenin or a control sequence. Cells were harvested for immunoblot analysis 72 h after siRNA transfection. Shown is β-catenin immunoblot analysis of 1%Triton, 1% Deoxycholate extracts from control and β-catenin siRNA treated MDCK cells. In β-catenin siRNA treated cells, β-catenin was decreased ∼70% (n = 3).(6.53 MB TIF)Click here for additional data file.Figure S2Phospho-β-catenin antibodies are specific. (A) P41/45 β-catenin antibody staining demonstrates cell membrane localisation in the absence but not in the presence of the competing phospho-peptide. MDCK cells were fixed and immunostained with P41/45-β-catenin antibodies preincubated with P41/45-β-catenin blocking peptide. Bottom panels show mitotic cells co-stained with DAPI showing the specific increase in cytoplasmic phospho-β-catenin in mitotic cells. Upper panels are confocal Z projections, lower panels are single confocal sections of immunostained MDCK cells, scale bar 20 µm. (B) P33/37/41 β-catenin antibody staining demonstrates cell membrane (arrow), centrosome (arrowhead), intracellular cluster (dashed arrow) as well as mid-body bridge (not shown), in the absence but not in the presence of the competing phosphopeptide. MDCK cells were fixed and immunostained with P33/37/41 β-catenin antibdies preincubated with P33/37/41 β-catenin blocking peptide. Shown are confocal Z projections of immunostained cells, scale bar 20 µm. Phospho-peptides contain the same sequence as the antigen used to raise phospho-β-catenin antibodies.(3.77 MB TIF)Click here for additional data file.Figure S332P-ATP followed by phospho-imaging, or by immunoblot with the indicated antibodies. (B) Autoradiograph of phosphorylated β-catenin from (A) showing incorporation of 32P phosphate followingphosphorylation by CK1α and CK1α+GSK3β but not GSK3β alone.Phospho-β-catenin antibodies recognise phosphorylated but not unphosphorylated β-catenin. (A) Immunoblot analysis of purified recombinant wild type β-catenin and S45A -β-catenin phosphorylated with CK1α alone, GSK3β alone and CK1α+GSK3β probed with β-catenin and phospho-β-catenin antibodies, as indicated. P41/45 phospho-β-catenin antibodies recognise β-catenin phosphorylated by CK1α alone and CK1α+GSK3β but not GSKβ alone; P33/37/41 phospho-β-catenin antibodies recognise β-catenin phosphorylated by CK1α+GSK3β but not CK1α or GSK3β alone. S45A β-catenin is not phosphorylated by CK1α and is therefore not recognised by P41/45 or P33/37/41-β-catenin antibodies. Full length mouse β-catenin , subcloned into pET-DUET as an Asc1/Not1 fragment, was expressed in E.coli BL21 (DE3) cells as an N-terminal His6 fusion protein was purified using nickel affinity and size exclusion chromatography. Active CK1α (a kind gift from Dr Jorge Allende) was expressed in E.coli BL21 (DE3) and part-purified as a His6 fusion protein by Ni-affinity chromatography. GSK3β with an N-terminal Flag epitope tag was subcloned into the baculovirus vector pBlueBac4 (Invitrogen) and recombinant protein produced in Sf9 cells. Flag-GSK3β was purified by affinity chromatography using anti-Flag-M2 agarose (Sigma) followed by size exclusion chromatography. 20 pmols purified recombinant His6-β-catenin or His6-β-catenin S45A mutant was phosphorylated in the presence of 1 mM ATP, 4 mM MgCl2, 150 mM NaCl, 20 mM Tris-HCl, pH 7.5 using recombinant His6-CK1α and Flag-GSK3β. Reactions were performed in a final volume of 20 µl at room temperature for 30 min, terminated by the addition of SDS-PAGE loading buffer, and separated by SDS-PAGE (4–12% Bis-Tris). Phosphate incorporation was verified by performing the reaction in the presence of 2 −μCi γ(1.68 MB TIF)Click here for additional data file.Figure S4Specificity of phospho-β-catenin antibodies (A) LIM2551 cells, containing mutated β-catenin that results in deletion of amino acids 5–72 (1.47 MB TIF)Click here for additional data file.Figure S5Detection of phosphorylated-β-catenin in colon cancer cells. Immunoblots of MDCK and colon cancer cell lysates demonstrate that the major band detected by P41/45- and P33/37/41-β-catenin antibodies co-migrates with β-catenin. Filters were probed with antibodies to the indicated proteins. Cells containing mutations in APC (LIM2537 and SW480) have detectable phospho-β-catenin. Cells containing β-catenin mutations at S45 (LIM1899 and HCT116) do not have detectable P41/45-β-catenin but are still phosphorylated at P33/37/41. LIM1215 cells contain a mutation at T41. Note that in LIM2551 cells, β-cateninΔ5–72 migrates faster by SDS-PAGE consistent with the size of the mutated protein and is not detected by P41/45 or P33/37/41. Cells treated with 30 mM LiCl do not show any change in P41/45-β-catenin, as expected, but show reduced P33/37/41-β-catenin as detected by P41/45- and P33/37/41-β-catenin antibodies, respectively. Shown are representatives of at least three separate experiments.(1.98 MB TIF)Click here for additional data file.Figure S6Axin is localised to cytoplasmic puncta and not at cell junctions or the ends of cell extensions in MDCK cells. MDCK cells were serum starved and untreated (control (A and C)) or treated with HGF (2 ng/ml) for 6 h (B and D), fixed and immunostained with axin and β-tubulin (A and B) and axin or APC (C and D) antibodies and co-stained with DAPI to visualise nuclei. Left panels are grey scale images of axin; Right panels are merged images, axin (green), β-tubulin/APC (red), nuclei (blue). Axin was not detected at the cell periphery or at the ends of microtubules in control or HGF stimulated cells. Scale bars 20 µm.(6.25 MB TIF)Click here for additional data file.Figure S7Phosphorylated β-catenin is localised in clusters to the ends of microtubules and is coincident with APC. MDCK cells were immunostained with antibodies to P41/45- (A) or P33/37/41-β-catenin (B) (green), APC (magenta) and β-tubulin (blue) and in (B) co-stained with TO-PRO-3 iodide to visualise nuclei (cyan). APC clusters and coincident phospho-β-catenin (white) are indicated with green arrowheads. Note that APC decorates the ends of microtubules; in tight colonies, the cell protrusions are not distinct and extend across the epithelial monolayer so it is sometimes not obvious that the APC clusters are at the ends of microtubules even when cells are costained with tubulin (see A). However, at the edges of colonies, the ends of microtubules are distinct and the APC clusters at cell protrusions are clearly visible.Shown are confocal sections, scale bar 20 µm.(8.56 MB TIF)Click here for additional data file.Figure S8Inhibition of β-catenin phosphorylation in MDCK cells. Lysates from MDCK cells treated with GSK3β and CK1α inhibitors and immunoblotted as indicated. (A) P33/37/41-β-catenin is reduced following treatment with all kinase inhibitors, but most strongly following inhibition with GSK3β inhibitors, LiCl and kenpaullone. (B) P41/45-β-catenin is reduced following treatment with D4476, and reduced slightly with IC261, but not by treatment with GSK3β inhibitors, LiCl and kenpaullone. C) P41/45-β-catenin remains localised in sub-populations at cell junctions (arrows) and in clusters coincident with APC (arrowheads) following treatment with GSK3β inhibitor, LiCl. Scale 10 µm.(3.21 MB TIF)Click here for additional data file.Figure S9Inhibition of CK1 results in loss of phospho-β-catenin at cell-cell contacts. MDCK cells were treated with vehicle control or D4476 (100 µM) for 1 h, fixed and immunostained with antibodies to P41/45 β-catenin or P33/37/41β-catenin (green), APC or E-cadherin (red), and co-stained with DAPI to visualise nuclei. (A) P41/45 β-catenin (green), APC (red), nuclei (blue). (B) P41/45 β-catenin (green), E-cadherin (red), nuclei (blue). (C) P33/37/41 β-catenin (green), APC (red), nuclei (blue). Phospho β-catenin cell contact staining, indicated by solid arrows, is lost in D4476 treated cells but co-incident APC staining, indicated by dashed arrows, and cytoplasmic staining in mitotic cells (*) remains after treatment. Shown are confocal sections, scale bars 20 µm.(7.60 MB TIF)Click here for additional data file.Figure S10Inhibition of protein phosphatases results in an increase in P33/37/41-β-catenin associated with APC. (A) MDCK cells were pre-treated with 30 mM NaCl or LiCl for 30 min, cells were washed 2× and incubated in DME+10% FCS containing vehicle control or okadaic acid (20 nM). Cells were harvested at T0, 5, 10 and 20 min and lysates immunoblotted with P33/37/41-anti rabbit-IR800 and reprobed with actin-anti-mouse-IR700 antibodies. The numbers below each lane reflect normalised densitometry values. Shown is a representative of at least 3 independent experiments. There is a modest increase in phosphorylated (P33/37/41)-β-catenin in the presence of the phosphatase inhibitor. (B) MDCK cells were pre-treated with 30 mM NaCl or LiCl for 30 min and cells were washed 2× and either fixed (T0) or incubated in DME+10%FCS without and with Calyculin (20 nM) for 20 min. Fixed cells were immunostained with P33/37/41-β-catenin (green) and APC (red) antibodies and costained with DAPI. P33/37/41-β-catenin coincident with APC clusters at cell protrusions is reduced with LiCl treament and is partially restored after LiCl is washed out. Incubation with phosphatase inhibitor, Calyculin, results in increased P33/37/41-β-catenin in clusters coincident with APC. Representative fields for each treatment are shown in left panels. Arrowheads indicate APC clusters. Scale bar 20 µm. Cells were scored for coincident P33/37/41-β-catenin and APC staining and plotted in right panel. Shown is average +/− SD from three independent experiments, >100 cells per treatment group. **P<0.02, ***P<0.005, ****P = 0.00005 by Students t test.(3.08 MB TIF)Click here for additional data file.Figure S11APC is required for phospho-β-catenin localisation to the ends of microtubules. MDCK cells transfected with control and APC siRNA were immunostained for P33/37/41 β-catenin (green), APC (magenta) and E-cadherin (blue) with grey scale images shown below and enlarged in insets. Arrowheads indicate clusters of phospho-β-catenin at cell extensions coincident with APC in control but not APC depleted cells. White arrows indicate intact junctional phospho-β-catenin; green arrows indicate concentrations of phospho-β-catenin coincident with E-cadherin. Scale bars 20 µ .(5.37 MB TIF)Click here for additional data file.


Here we show that CR induces a two-fold increase SIRT1 expression in the intestine of rodents and that ectopic induction of SIRT1 in a β-catenin-driven mouse model of colon cancer significantly reduces tumor formation, proliferation, and animal morbidity in the absence of CR. We show that SIRT1 deacetylates β-catenin and suppresses its ability to activate transcription and drive cell proliferation. Moreover, SIRT1 promotes cytoplasmic localization of the otherwise nuclear-localized oncogenic form of β-catenin. Consistent with this, a significant inverse correlation was found between the presence of nuclear SIRT1 and the oncogenic form of β−catenin in 81 human colon tumor specimens analyzed. Taken together, these observations show that SIRT1 suppresses intestinal tumor formation in vivo and raise the prospect that therapies targeting SIRT1 may be of clinical use in β−catenin-driven malignancies.Numerous longevity genes have been discovered in model organisms and altering their function results in prolonged lifespan. In mammals, some have speculated that any health benefits derived from manipulating these same pathways might be offset by increased cancer risk on account of their propensity to boost cell survival. The Sir2/SIRT1 family of NAD SIR2 gene is proposed to mediate the health benefits of CR SIR2, is induced by CR in multiple tissues of mammals, and has been shown to ameliorate degenerative diseases associated with aging, such as neurodegeneration and metabolic decline in vitro as to whether SIRT1 will be found to act as an oncogene or as a tumor suppressor but to date there have been no in vivo studies that address this question. On the one hand, SIRT1 is upregulated in tumors and cancer cells lacking the tumor suppressor gene, HIC1 in vivo. On the other hand, SIRT1 can be pro-apoptotic in vivo. Moreover, some have argued that mammalian longevity genes that delay age-related atrophic diseases may conversely predispose humans to a higher incidence of cancer due to their anti-apoptotic function Cancer is the second leading cause of age-related mortality in humans. Calorie restriction extends lifespan in all organisms tested and in mammals exerts strong tumor suppressive effects min/+ model of colon cancer for a variety of reasons: it physiologically recapitulates the early events of colon cancer in humans, the mechanism of tumorigenesis is well characterized, and CR has previously been shown to reduce the rate of tumorigenesis in this model min/+ mouse contains a germline mutation in the adenomatous polyposis coli (APC) tumor suppressor gene We chose to test the effect of SIRT1 in the APCβ-catenin is the central effector in the canonical Wnt signaling pathway that controls stem cell maintenance, development and carcinogenesis min/+ mouse model and inhibits colon cancer growth. We provide substantial evidence that the anti-tumorigenic effects of SIRT1 depend on its deacetylase activity and are mediated through inhibition of β-catenin. These findings identify a tumor suppressive function for SIRT1, provide mechanistic insight, and suggest a therapeutic role for SIRT1 deacetylase activators in colon cancer.We report here that SIRT1 suppresses intestinal tumorigenesis in the APCad lib-fed controls  which overexpress SIRT1 specifically in the gut villi (referred to as SIRT1ΔSTOP). The SIRT1 levels in the gut of SIRT1ΔSTOP mice were approximately 7-fold (Earlier studies have shown a dramatic tumor suppressive effect of CR but the molecular mechanism(s) are currently unknown. We observed that rats on a CR diet have ∼2-fold higher levels of SIRT1 in the gut epithelium relative to controls . To testcontrols . SIRT1 ty 7-fold  and the y 7-fold .min/+, SIRT1STOP control mice that did not overexpress SIRT1 (referred to as SIRT1STOP) showed the typical signs of tumor morbidity at 16 weeks of age, as evidenced by overt anemia and cachexia, whereas APCmin/+ mice overexpressing SIRT1 (SIRT1ΔSTOP) displayed no overt signs of tumor associated morbidity  and Ki-67 staining, demonstrating that there was a decrease in adenoma proliferation  in different human colon cancer cell lines whose growth is driven by constitutively active β-catenin (HCT116 and DLD1). A human colon cancer cell line in which β-catenin is inactive (RKO) served as a negative control. Increased SIRT1 expression greatly reduced proliferation in both colon cancer cell lines with constitutively active β-catenin but not in the β-catenin-inactive cell line . Knockdown of β-catenin dramatically reduced reporter activity, demonstrating the dependence of the reporter on endogenous β-catenin activity . When β-catenin expression was scored in these same colon cancers, a highly significant inverse correlation between the level of SIRT1 expression and nuclear β-catenin localization became apparent  . Collectmin/+ animals mimics the tumor suppressive effects of CR on this colon cancer model. These observations are consistent with a recent in vitro study that implicates SIRT1 as a nutrient sensitive growth suppressor in vivoin vivo evidence that overexpression of SIRT1 at physiologically relevant levels, can suppress tumor formation and growth.Here we describe a physiologically relevant tumor suppressive role for SIRT1 in colon cancer formation and growth. We observed that SIRT1 expression in the normal intestine occurs specifically in the enterocytes, the precursor cells that undergo neoplastic transformation in colon cancers and that SIRT1 is upregulated in rodent intestines in response to CR. We show that overexpression of SIRT1 reduces proliferation in colon cancer cell lines and that overexpressing SIRT1 in the enterocytes of APCmin/+ model and a variety of human tumors. We find that SIRT1 overexpression inhibits the growth of colon cancer cells dependent on β-catenin activity, suppresses the localization of β-catenin to the nucleus, and significantly attenuates its ability to activate transcription. These effects were not observed in the SIRT1-HY mutant demonstrating that SIRT1 deacetylase activity is required, and raising the possibility that SIRT1 directly targeted β-catenin for deacetylation.In this study, we also present evidence that SIRT1 interacts with and suppresses β-catenin, the transcription factor that drives tumors in the APCin vivo.Previous studies have shown that β-catenin is acetylated by p300/CBP and the acetylated form of the protein has increased transcriptional activity. This finding implies that the putative deacetylase that counteract p300/CBP would be useful as a cancer therapeutic target Together, our data sheds light on the ability of SIRT1 to inhibit β-catenin activity and provides mechanistic insight into the anti-tumorigenic effects of SIRT1 in a well characterized colon cancer model. Given that SIRT1 was discovered as a homolog of a longevity gene, it is interesting to note the growing evidence for a link between Wnt/β-catenin signaling and age-associated diseases. β-catenin has been linked to other age-associated malignancies such as melanoma, multiple myeloma, and prostate cancer. There is also the recent discovery that upregulation of Wnt/β-catenin signaling accelerates aging in the mouse In summary, using biochemistry, mouse genetics, and clinical tumor specimens we have found that SIRT1, a diet-responsive gene, is a regulator of β-catenin and has a tumor suppressive function. We conclude that mammalian longevity genes with anti-apoptotic functions, surprisingly do not necessarily lead to increased tumorigenesis. In fact, we find the opposite is likely the case for SIRT1. These studies begin to answer an important biological question regarding the function of a key longevity gene in cancer development and growth and suggest a previously unidentified therapeutic potential for SIRT1 activators in cancer.loxP flanked transcriptional STOP element was inserted between a CAGGS promoter and the SIRT1 cDNA. This construct was targeted into the mouse Collagen A1 locus using flp recombinase-mediated genomic integration as described previously (1). MES cells carrying a single copy of the SIRT1STOP construct were identified by resistance to the antibiotic marker hygromycin and Southern blotting. PCR primers and construct maps are available upon request. Two clones were injected into blastocysts and both generated pups, ∼90% of which displayed germ-line transmission. Tumor bearing mice that were analyzed had been backcrossed at least four generations into C57/BL6. APCmin/+ and Villin-Cre transgenic mice strains were obtained in the C57/BL6 background from Jackson Labs . SirT1STOP animals were backcrossed two generations into C57BL/6 mice before crossing to APCmin/+ to generate SirT1STOP; APCmin/+ double transgenics. These animals were bred to Villin-Cre transgenic mice to generate a cohort of SirT1ΔSTOP; Vil-Cre; APCmin/+ animals. Animals were maintained at Harvard Medical School and experiments were approved by the Animal Care Committee of Harvard Medical School.A Cre-inducible SIRT1 expression construct was generated in which a Male Fischer-344 (F344) rats were bred and reared in a vivarium at the Gerontology Research Center . From weaning (2 Wks), the rats were housed individually in standard plastic cages with beta chip wood bedding. Control animals were fed a NIH-31 standard diet ad libitum (AL). At 1 month of age the calorie restricted (CR) animals were provided a vitamin and mineral fortified version of the same diet at a level of 40% less food (by weight) than AL rats consumed during the previous week. Filtered and acidified water was available ad libitum for both groups. The vivarium was maintained at a temperature of 25°C with relative humidity at 50% on a 12/12-hour light/dark cycle (lights on at 6:00 a.m.) All animals were 6 months of age and sacrificed between 9:00 and 11:00 a.m. The intestine was quickly removed and thoroughly flushed with ice cold PBS and placed into liquid nitrogen then stored at −80°C until processed for Western blotting using standard procedures.For gross tumor analysis, the entire intestine was excised immediately after sacrifice, opened lengthwise and washed with cold phosphate-buffered saline (PBS) while pinned down a solid support. Adenomas larger than 0.5mm from the proximal , distal , and middle  small intestine as well as the colon were scored. Intestines were prepared using the Swiss roll method by rotating them around a glass pipette tip. Tissues were fixed and embedded in paraffin using standard histology protocol. Precise tumor size was scored microscopically on hematoxylin/eosin stained of mouse intestines using a microscope with an eyepiece micrometer. Immunohistochemical analysis of rodent tissue was performed with rabbit anti-SIRT1 antibody , rabbit anti-β catenin (abcam #2982), mouse anti-β-catenin  and rat anti-mouse Ki-67 (Dako).2O2 (15 min) to block endogenous peroxidase, then incubated with 10% normal goat serum  in PBS (10 min), followed by 10 min incubation in serum free protein block . Primary antibody against β-catenin  (dilution 1∶400) or SIRT1  (dilution 1∶100) was applied for 1 hour at room temperature. Secondary antibody (BioGenex) (20 min), and then streptavidin peroxidase conjugate (BioGenex) were applied (20 min). Sections were visualized by diaminobenzidine (DAB) (2 min) and methyl-green counterstain. Nuclear expression was recorded as either no expression, weak expression, or moderate/strong expression. Positivity in nucleus was defined as moderate/strong expression. All β-catenin-stained TMA slides were interpreted by a pathologist (S.O.) blinded from any other clinical and laboratory data. All SIRT1-stained TMA slides were interpreted by a pathologist (R.F.) blinded from any other clinical and laboratory data. For statistical analysis, chi-square test was performed for categorical data using the SAS software program . The p-value was two-sided, and statistical significance was set at p ≤ 0.05.Tissue microarrays (TMAs) were constructed as previously described ΔHY, pcDNA-HA-S33Y-β-catenin and pBABE-Puro-S33Y- β-catenin have been described before. SIRT1 RNAi plasmids were constructed by cloning the sequences into the pSUPER.retro plasmid . One TOPFLASH plasmid was purchased from Upstate Biotechnology  while the second was generated by cloning the tandem TCF binding sites and TA-promoter from SUPER8xTOPFLASH  into the Luciferase-PEST plasmid pGL4.15 .pcDNA3-FLAG-SIRT1, pBABE-Puro-hSir2 (SIRT1), pBABE-Puro-SIRT12 (5% v/v) at 37°C. For over-expression experiments, plasmids were transfected by the Fugene 6 method (Roche). For stable cell line generation, DLD1 cells were selected in hygromycin for two weeks and single colonies were picked and expanded. For retroviral production, 293T cells were transfected with the overexpression or RNAi plasmids simultaneously with packaging plasmids gag-pol and VSV-G or pCL-ampho. The media containing the progeny virus released for the 293T cells was collected and used to infect the cells for 3–6 hours in the presence of 8 µg/ml polybrene . The medium was changed and cells were incubated for additional 24–48 hour incubation. They were selected with puromycin (Sigma Aldrich) for 24–48 hours and then trypsinized and seeded for experiments.293T, LN-CAP, DLD1, HCT116 and RKO cells were maintained in the recommended tissue culture media (American Type Culture Collection (ATCC), Manassas, VA) and grown in a humidified incubator containing COFor immunocytochemistry, cells were plated on 8 mm six well Teflon printed slides (Electron Microscopy Sciences). Twenty four hours after plating, cells were fixed by incubation with 4% Paraformaldehyde for 30 minutes. Fixed cells were then incubated with primary antibodies to SIRT1  and active β-catenin  (dilution 1∶100) for 2 hours followed by incubation with secondary antibodies  (dilution 1∶400). Cells were incubated with 10 µg/ml Hoechst, washed and mounted with a coverslip. At least 20 cells were scored per experiment and image capture was performed using an Olympus BX50 microscope.g for 10 min at 4°C, and the cleared supernatants were employed for immunoprecipitation. Lysates were immunoprecipitated (2 hr), washed 4 times with NP-40 buffer and were processed by Western blotting.Cell extracts analyzed directly by Western blotting were prepared by cell lysis in 1× SDS loading buffer followed by boiling and Western analysis. Cell extracts for immunoprecipitation were prepared by resuspending phosphate-buffered saline-washed cell pellets in 1 ml of Nonidet P-40 (NP-40) extraction buffer  supplemented with EDTA-free protease inhibitor cocktail tablets (Roche) with 10 mM Nicotinamide (NAA) and 5 µM Trichostatin-A (TSA). Following incubation on ice for 30 minutes, non-extractable material was removed by centrifugation at 17,000 DLD1, HCT116 and RKO cell lines infected with the appropriate construct were seeded in 24 well plates at a density of 10,000 cells. Cells were trypsinized and analyzed by Coulter counting at given time points.Luciferase assay was done as instructed by the dual luciferase reporter assay system or firefly luciferase assay system .http://rsb.info.nih.gov/ij/)For expression studies, animal intestines were flushed with cold PBS and either homogenized whole or scraped to enrich for enterocytes. Protein extracts were prepared by dounce homogenization in standard lysis buffer, subjected to SDS-PAGE and transferred onto PVDF membranes. Membranes were immunoblotted using rabbit polyclonal antibody to SIRT1  and rabbit polyclonal antibody to β-actin (Abcam cat #8226). Densitometric analysis was performed on scanned images of blots using ImageJ software  APCmin/+ mice overexpressing SIRT1 (SIRT1ΔSTOP) show less anemia than their non SIRT1 overexpressing age matched counterparts (SIRT1STOP) as reflected by the color of their paws. (B) Graph depicting average body weight of mice at time of sacrifice (n = at least 4 per group). Error bars represent average±S.D.APC(2.31 MB TIF)Click here for additional data file.


The complexity of wnt signaling likely stems from two sources: multiple pathways emanating from frizzled receptors in response to wnt binding, and modulation of those pathways and target gene responsiveness by context-dependent signals downstream of growth factor and matrix receptors. Both rac1 and c-jun have recently been implicated in wnt signaling, however their upstream activators have not been identified.Here we identify the adapter protein Grb2, which is itself an integrator of multiple signaling pathways, as a modifier of β-catenin-dependent wnt signaling. Grb2 synergizes with wnt3A, constitutively active (CA) LRP6, Dvl2 or CA-β-catenin to drive a LEF/TCF-responsive reporter, and dominant negative (DN) Grb2 or siRNA to Grb2 block wnt3A-mediated reporter activity. MMP9 is a target of β-catenin-dependent wnt signaling, and an MMP9 promoter reporter is also responsive to signals downstream of Grb2. Both a jnk inhibitor and DN-c-jun block transcriptional activation downstream of Dvl2 and Grb2, as does DN-rac1. Integrin ligation by collagen also synergizes with wnt signaling as does overexpression of Focal Adhesion Kinase (FAK), and this is blocked by DN-Grb2.These data suggest that integrin ligation and FAK activation synergize with wnt signaling through a Grb2-rac-jnk-c-jun pathway, providing a context-dependent mechanism for modulation of wnt signaling. The wnt signaling pathway regulates a variety of cellular processes including fate specification, establishment of polarity, proliferation, migration and tissue morphogenesis 2+ and wnt/ROR2 pathways The basics of canonical wnt signaling are well known: in response to wnt signaling β-catenin is stabilized, enters the nucleus, and acts as a transcriptional co-activator by interacting with LEF/TCF transcription factors bound to specific sites in the promoters and enhancers of wnt target genes. In the absence of wnt signaling, cytoplasmic β-catenin is recruited to a complex containing axin, APC and GSK-3β, which is then able to phosphorylate β-catenin, triggering its ubiquitin-dependent degradation. Wnt binding to frizzled (Fz) receptors and low-density lipoprotein related protein (LRP 5/6) co-receptors triggers a disheveled (Dvl)-dependent disaggregation of the APC-axin-GSK-3β complex, resulting in accumulation of β-catenin and its subsequent translocation into the nucleus in vitro, wnt induction of MMP expression was considerably stronger in T cells migrating through collagen, than in cells cultured on plastic or gelatin (denatured collagen). These data suggest that wnt signaling in T cells may be affected by integrin-mediated cell-matrix interactions, consistent with a previous finding of dwnt4/focal adhesion kinase (FAK) interaction in drosophila Our previous work demonstrated that wnt signaling can induce matrix metalloproteinase (MMP) expression in T cells and regulate their transmigration across an endothelial monolayer and its underlying basement membrane, into subjacent tissues A common element downstream of integrins and growth factor receptors is the adapter protein growth factor receptor-bound-2 (Grb2), which, through its SH2 and SH3 domains, coordinates multiple inputs to regulate activation of several signaling cascades Anti-FLAG and anti-tubulin were from Sigma . Anti-HA (Clone Y-11) was from Santa Cruz Biotechnology . Anti-β-catenin was from BD Transduction Labs . Anti Grb2 was from Cell Signaling, and anti-dvl2 was from Santa Cruz. The JNK inhibitor (SP600125) was from Calbiochem . Human FGF2 (bFGF) was from Invitrogen .All three human Dishevelled isoforms were gifts from Dr. Misha Semenov . Dishevelled cDNA was cloned in frame with a C-terminal FLAG tag into pcDNA 3.1+ . Human Grb2 was cloned from cDNA and subcloned in frame with an N-terminal HA tag into pcDNA 3.1+. SuperTOPflash and super FOPflash were gifts from Dr. Randall Moon . The wnt3A expression plasmid was from Dr. Karl Willert . Wnt3a cDNA was subcloned into pcDNA3.1+. N-terminally truncated LRP6 was kindly provided by Dr. Anthony Brown . S33Y β-catenin was a gift from Dr. Avri Ben-Ze'ev . The β1-integrin cytoplasmic domain fused to Tac was a gift of Dr. Mark Ginsberg . An 800 bp fragment of the human MMP9 promoter has been previously described Human embryonic kidney cells (HEK293 cells) and monkey COS7 cells were maintained in Dulbecco's modified Eagle Medium (DMEM)  supplemented with 10% fetal bovine serum (FBS) and 1∶100 Penicillin/Streptomycin (P/S) . Wnt3a-conditioned medium was collected from control or Wnt3a-expressing L-cells (ATCC) cultured for 7 days.© . As COS7 have very low endogenous levels of LEF/TCF proteins all experiments using these cells also included transfection with TCF1E. Cells were seeded the night before transfection at 3×105 cells per well into C6-well plastic dishes pre-coated with gelatin . On the next day, 2.5 µL of Lipofectamine 2000© was diluted in 100 µL of OptiMEM  and left to incubate for 5 minutes at room temperature. 1–1.25 µg of DNA was diluted in 100 µL of OptiMEM and the two mixtures were combined and left to incubate for 20 min at RT. In all experiments involving sTF it was used at 250 ng. SP5-Luc ©/OptiMEM were prepared. Cells were washed 1X with HBSS and 2 mL of DMEM without serum or P/S was added. 200 µL of the transfection mix was added per well and the cells were incubated for 1 hr at 37°C. Cells were then washed 1X with HBSS and replaced with 10% DMEM with no antibiotics. Transfected cells were then incubated for various times until harvesting. Cells transfected for immunoprecipitation were seeded the night before transfection in 10 cm tissue culture dishes at 2×106 per plate. The next day, the cells were transfected with 5 µg of total DNA. For siRNA experiments cells were transfected as above with siRNA duplexes obtained from Invitrogen. Knockdown was assayed at 24 hours by Western blot.HEK293 and COS7 cells were transfected using Lipofectamine 2000Luciferase enzymatic activity in the cell lysates was measured using a luminometer . Aliquots were assayed for total protein content using a BCA protein assay . Results are presented as Relative Light Units per µg protein (RLUs/µg protein).5′-TATGATTCCCGGTCCTCCTGGTC-3′; Lower strand: 5′-TGGCTCCTGCTCCTTTCTCTGTTC-3′HEK293 cells were transfected as above and harvested for qPCR as previously described For immunoprecipitations, cells were harvested by centrifugation and washed one time with DPBS on ice. Cells were lysed on ice for 15 minutes, with RIPA buffer  containing 1∶100 protease and phosphatase inhibitors . The lysates were vortexed, cleared by centrifugation and either frozen at −80°C or used immediately for immunoprecipitation. 500–600 µg of protein was used for immunoprecipitation. Cell lysates were incubated with 2 µg of anti-FLAG  at 4°C overnight, and then further incubated with protein-G sepharose  with rocking at RT for 2 hrs. Beads were then centrifuged, washed once with 500 µL of RIPA + protease/phosphatase inhibitors, 3 times with 1 mL of RIPA with no inhibitors, and then boiled in sample buffer. Whole cell lysates were prepared before immunoprecipitation and represent the input fraction. Boiled samples were loaded and run on an SDS-PAGE precast gels at 100 V for 1–2 hrs , blotted onto PVDF membranes  and then blocked with either 5% nonfat milk or 5% BSA diluted in wash buffer (0.1% tween-20 in TBS). After incubations with primary and secondary antibodies at 4°C blots were developed using the ECL chemiluminescent detection kit .Human embryonic kidney 293T cells were plated on glass coverslips and fixed with 4% paraformaldehyde for 20′ at 4°C followed by permeabilization with 0.5% Triton X-100 for 10′ at room temperature. Non-specific sites were blocked with 5% goat serum in PBS for 15′ at 37°C. Primary antibodies, Grb2 Ab (1∶1000) and Dvl2 Ab (1∶200) were diluted in PBS and co-hybridized to the fixed, permeabilized cells for 1 hr at room temperature. Cells were then washed with PBS three times and incubated with goat anti-mouse Alexa488 and goat anti-rabbit Alexa594 sera at (each at 1∶1000) for 30 min. Cells were then washed three times, mounted on slides and imaged.http://scansite.mit.edu/) and confirmed the presence of this site in all of them. In addition, we also identified a second proline-rich region (PRR) in the N-terminal region of Dvl2, which is absent in Dvl1 and Dvl3  and showed that this is important in canonical wnt signalingand Dvl3 . These Pin vivo we used co-immunoprecipitation. We co-expressed FLAG-tagged Dvl2 with HA-tagged Grb2 and lysates were immunoprecipitated with an anti-FLAG antibody. Blotting with an HA-antibody confirmed specific pull-down of Grb2 by Dvl2  and central  proline-rich regions  is a target of β-catenin-dependent wnt signaling in T cells To test whether Grb2 is required for β-catenin-dependent wnt signaling we utilized siRNA-mediated gene targeting. However, we were unable to identify sequences that generated complete knockdown of Grb2 expression, achieving only a 50% reduction at 24 hours . DespiteGrb2 contains two SH3 domains, flanking an internal SH2 domain. Previously, it has been shown that specific point mutations in the SH3 and SH2 domains of Grb2 lead to a dominant negative protein Our data showing that Grb2 strongly synergizes with CA-β-catenin suggest that it operates either downstream of, or in parallel with, β-catenin, rather than acting to stabilize the protein. To test this hypothesis cells were transfected with Dvl2 or Grb2 and extracts were blotted for β-catenin protein. While Dvl2 effectively stabilized β-catenin, Grb2, even at high doses, had no effect on β-catenin levels . SeveralFinally, we wished to determine what signals upstream of Grb2 feed into the wnt pathway. Our previous studies had shown that wnt signaling in T cells was optimal when the cells were migrating through a collagen matrix Integrin signaling is mediated largely through two receptor-associated kinases, integrin-linked kinase (ILK) and focal adhesion kinase (FAK) Grb2 is also an important mediator downstream of growth factor receptors. As an initial test of whether serum factors may provide input to the wnt pathway we transfected cells with sTF and either Dvl2 alone or Dvl2 plus Grb2 and cultured the cells in 10% or 2% serum, or serum-free medium. Dvl2-mediated sTF activity was significantly and dose-dependently reduced in lowered serum , and thiin silico analysis suggested that the proline-rich domains of Dvl2 might be ligands for the SH3 domains of Grb2 and immunoprecipitation analysis confirmed this interaction. Interestingly, although Grb2 appears to interact with PRR1 and PRR2, our data suggest that only the interaction with PRR2 is absolutely required for functional synergy between the two (Transcription dependent upon β-catenin is increasingly being seen as the output of numerous pathways acting in synergy with the “canonical” wnt-Fz/LRP-dvl pathway. Our data reveal a link between the adaptor protein Grb2, which acts downstream of growth factor receptors and integrins, and wnt signaling operating through Dvl2. Grb2 is a multi-domain protein that functionally integrates signals from numerous cell-surface receptors and its absence leads to early embryonic lethality  the two .A previous report Our previous study in T cells demonstrated that wnt signaling was considerably enhanced in cells migrating through collagen gels Our finding that DN-Rac1 blocks synergy between Dvl2 and Grb2 is entirely consistent with a previous report that Rac1 forms a complex with β-catenin and Jnk in the cytoplasm and is required for Jnk phosphorylation of β-catenin and its subsequent transport into the nucleus In conjunction with published studies, our data now help to provide a clearer picture of how wnt signals are coordinated with adhesion and growth factor receptors . Wnt binIn summary, we have identified a role for Grb2 in coordinating signals downstream of FAK and growth factor receptors with the wnt pathway. Grb2 thus provides a way for cells to tune wnt signaling depending on the extracellular context.


Aberrant Wnt-signaling caused by mutants of β-catenin, a key regulator of the canonical Wnt-signaling pathway, is frequently detected in cancer. Only recently, it was suggested that in hepatocellular carcinoma (HCC) the expression of the target gene glutamine synthetase (GS) is a highly reliable marker for the identification of β-catenin mutations. In order to prove this hypothesis, 52 samples from human hepatocellular carcinomas were analysed for the activation of β-catenin and the expression of GS. In total, 45 samples stained positive for cytoplasmic/nuclear β-catenin. A strong correlation between expression of GS and activated β-catenin (100% of nuclear and 84% of cytosolic) was found. However, among 35 GS positive tumors that were analysed for β-catenin mutations no mutations were detected in 25 GS-positive carcinomas although 24 out of the 25 carcinomas exhibited at least abnormal expression of β-catenin. Since the mutational analysis identified 9 different point mutations of the β-catenin gene including the rare mutation H36P and the yet unknown mutation P44A it was asked whether these mutations may differently effect β-catenin target genes. Therefore, expression plasmids for different mutations were constructed and cotransfected with the TOP-flash luciferase reporter and a reporter carrying the GS-5'-enhancer. The experiments confirmed that there are differences between different β-catenin target sequences and different β-catenin mutations. In addition, the failure that the endogenous expression of GS in GS-negative cells was not induced by the transient transfection experiment indicated that the effect of β-catenin on the GS-5'-enhancer is only one aspect of gene activation induced by β-catenin. Hepatocellular carcinoma (HCC) is the most frequent cancer worldwide . Duriet al. demonstrated that GS expression may enhance the metastatic potential in HCC, and that GS immunostaining identifies HCC patients with a high risk for disease recurrence after curative hepatectomy [In differentiated, non-proliferating cells, β-catenin is associated with membrane bound E-cadherin  and non-atectomy . In the atectomy . With reatectomy -22 as weatectomy ,24. ReceIn a first series of experiments 52 human tumor samples were analysed for the expression of GS and β-catenin. Immunohistochemistry identified β-catenin positive nuclei in 13 specimens . Therefore, DNA was extracted from samples, amplified by PCR and sequenced. It was possible to amplify DNA from 40 samples out of the 52 specimens that were investigated by immunohistochemistry. The analysis revealed 9 different point mutations of the CTNNB1 gene in 10 (25%) patients . However, only one of the samples expressed cytosolic β-catenin that interestingly also expressed GS. The other tumor sample showed membranous β-catenin and no obvious GS expression.Since activation of β-catenin can also be caused by mutations in other genes of the Wnt-signaling pathway, the APC-, the GSK-3β- and the β-catenin-binding domains of the HindIII/EcoRV-fragment from the rat GS gene encompassing the DNA sequence from position -2516 to -2146 upstream from the transcriptional start point that includes the 5'-enhancer complex [Since we wondered whether the mutations of β-catenin detected in samples of human hepatocellular carcinomas are directly responsible for the activation of Wnt-target genes and especially for the expression of GS, plasmids were developed in order to express the mutated forms of β-Catenin in transfected cells. Therefore, we constructed expression plasmids, including ones for the rare mutation H36P and the non-described mutation P44A as well as expression plasmids for S45F and S45P. Additionally, reporter gene expression in the presence of these new constructs was compared to expression in the presence of plasmids carrying a truncated β-catenin, lacking the N-terminal phosphorylation sites ('pBatem') and a β-catenin with all four phosphorylation sites mutated to alanine, designated 'pCS2' . In order to normalize for endogeneous activity of Wnt-signaling the control plasmid 'bCATa' was used. Firstly, the reporter genes were cotransfected into HuH7 cells together with the 'TOP-flash' plasmid that harbours six LEF-1/TCF-binding sites. As control the same plasmids were cotransfected with Fop-flash that is equal to 'TOP-flash' but does not contain the LEF-1/TCF-binding sites. Forty eight hours after transfection cells were harvested and luciferase activity was determined. The factor of enhancement was determined by comparing 'TOP-flash' expression with expression from 'FOP-flash' for each of the different expression plasmids used. The result of the experiment is presented in Fig.  complex . HuH7 ceSince reporter gene experiments clearly demonstrated that the GS-5' enhancer is a target for enhancement of expression mediated by mutated β-catenin, it was asked how the expression of endogenous GS is influenced by β-catenin mutants. Therefore, the expression plasmid for β-catenin with S45F that proofed to be most effective in reporter gene experiments was transfected into HuH7 cells and GS activity was determined. However, we were not able to detect any enhanced expression of GS between 24 and 120 hours after transfection (data not shown). The same result was obtained with cells from other lines as FAO or primary rat hepatocytes, no matter whether overall GS-activity of the transfected culture was determined or single cell determination of GS expression using specific antibodies was employed (data not shown).The nuclear and/or cytoplasmic stabilization of β-catenin is recognized as a hallmark of an altered Wnt/β-catenin pathway . At the With regard to mutations of the Axin1 gene we have identified a 3 bp deletion affecting the tyrosine at position 97 within the APC-binding site of Axin1 in two out of 18 analysed HCC. Only one of these two samples expressed cytosolic β-catenin and GS and the other did not express β-catenin nor GS. Therefore, loss of Axin1 function does not necessarily cause aberrant expression of β-catenin and GS in agreement with Zucman-Rossi et al. .In conclusion, the diagnostic value of GS expression for the detection of β-catenin mutations or even for Wnt-signaling is doubtful, since tumors can be detected that express GS even in the absence of aberrant β-catenin expression and not every tumor with cytosolic β-catenin expresses GS.One important question of the work presented was to analyse how mutations of β-catenin are able to influence expression of GS. The cell line HuH7 was chosen for transfection experiments, since it had a low Wnt-signaling background activity compared with cells from the lines FAO or HepG2 (data not shown). Reporter genes with the GS-5'-enhancer and the 'TOP-flash' reporter plasmid were transfected together with expression plasmids for different mutated β-catenins into HuH7 cells. The strongest factor of enhancement (~17 fold with the GS-5' enhancer and ~22 fold with 'TOP-flash') was observed in the presence of the mutation S45F. Serine 45 is known as the priming position since its phosphorylation by casein kinase 1 (CKI1) is necessary for the subsequent phosphorylation of T41, S37 und S33 by glycogen synthase kinase 3β (GSK3β) that finally leads to ubiquitinylation and degradation of β-catenin by the proteasome. Therefore, from this point of view it was not surprising that a mutation of this position caused the highest effect. However, S45P affecting the same priming position was less effective in both, 'TOP-flash' and the GS-5'-reporter. Interestingly, this mutation did not result in nuclear staining for β-catenin .We analysed 52 samples of HCC from patients who had undergone operations at the Uniklinik Leipzig between 1999 and 2005. All patients provided written informed consent according to the German laws as confirmed by the local committee. After surgery the material was frozen in liquid nitrogen and kept at -80°C until use. Sections from each specimen were examined by a pathologist and classified as HCC.The surgically resected HCC specimens were fixed in 10% buffered paraformaldehyde and embedded in paraffin according to standard protocols. Analysis of β-catenin and GS expression was performed on serial sections (4 μm) from each tumor. After deparaffinization and rehydratation antigen retrieval was applied by microwaving (3 × 5 min) in citrate buffer . The endogenous peroxidase was destroyed by hydrogen peroxide ) and incubated for 10 min. Slides were then immersed in a solution with 0.05% avidin and 0.01% biotin (10 min) and finally incubated for 30 min in 5% goat serum  in order to block unspecific signals. As primary antibody a β-catenin monoclonal antibody  was used at a dilution of 1:90, incubated overnight at 4°C. After washing with TBS, the slides were incubated with a goat anti-mouse IgG-Fc-Biotin  antibody diluted 1:1000, washed again in TBS, and incubated with POD-conjugated Extravidin  for 30 min. Subsequently, a POD staining with 3,3'-Diaminobenziden (DAB)  for 7 min according to manufacturer's instructions was performed. After counterstaining with aqueous hematoxylin the dehydrated slides were covered in Roti-Histokitt .Detection of glutamine synthetase was performed with the following modifications to the protocol for β-catenin: The deparaffinized and rehydrated sections were treated with 3% hydrogen peroxide and 10% goat serum. The anti GS monoclonal antibody  used was diluted 1:1000 and incubated overnight at 4°C. The secondary POD-conjugated goat anti mouse IgG-FAB antibody  was applied at a dilution of 1:150 and incubated for 90 min. After washing the antibody reaction was visualized with DAB as substrate. A counterstaining was performed with aqueous hematoxylin before dehydration and covering using the Roti-Histokitt.®Tissue Kit, QIAGEN, Düsseldorf, Germany). The DNA sequence positions of the β-catenin and Axin1 genes were derived from NCBI reference sequences NM001904 and AF009674, respectively. To screen for mutations in the β-catenin gene (codons 5–79 of exon 3) and the Axin1 gene , genomic DNA was amplified by PCR using the following primer pair for β-catenin: 'βCAT exon 3 up' (5'-CAAT GGG TCA TAT CAC AGA TTC TT-3')/'βCAT exon 3 down' (5'-TCT CTT TTC TTC ACC ACA ACA TTT-3') and 8 pairs for the Axin1 gene: 'Axin 2/1 for' (5'-TGG TTT CAA CAG GAC AGA TTG-3') and 'Axin 2/1 rev' (5'-TCC CGG AGC AGA AAC TGT AG-3'); 'Axin 2/2 for' (5'-TAT CCA AGA GCA GGG TTT CC-3') and 'Axin 2/2 rev' (5'-GGC TTA TCC CAT CTT GGT CA-3'); 'Axin 2/3 for' (5'-GAG ACT TCG ACG GCC ACT C-3') and 'Axin 2/3 rev' (5'-TTC TCC TCG TTC GAG TCA CA-3'); 'Axin 2/4 for' (5'-GAG TCA CTG CAT TCC CTG CT-3') and 'Axin 2/4 rev' (5'-CTG GTA AAA CAT GGC AGG AT-3'); 'Axin 2/5 for' (5'-CTG GCG AGA GCC ATC TAC C-3') and 'Axin 2/5 rev' (5'-TAC AGA CTT TGG GGC TCT CC-3'); 'Axin 4 for' (5'-GGG GCC AGG AGC TCT ATT-3') and 'Axin 4 rev' (5'-TAG AGG TAA GCC TGG CTC GT-3'); 'Axin 5 for' (5'-CGC TCT TTC CCT TCA CAA AG-3') and 'Axin 5rev' (5'-CCC CAT GAA GAA CAT CAG GA-3'); 'Axin 6/1 for' (5'-TGC TTT TTG TTT TCC CCA AG-3') and 'Axin 6/1 rev' (5'-CAG GAT GCT CTC AGG GTT CT-3').For the isolation of DNA, 25 mg tumor tissue were used according to manufacturer's instructions  using 100 ng of genomic DNA and 15 pmol of each primer. Cycle conditions were: 94°C for 30 s for denaturation and 55°C for 40 s for hybridization. Elongation was done at 72°C for 45 s. The enzyme was initially activated by denaturation at 95°C for 3 min and final extension was performed at 72°C for 10 min. The PCR products were checked on standard agarose gels and purified before they were sequenced.Cycle sequencing was performed at the IZKF Core Unit Leipzig using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems). Sequencing products were applied to an ABI prism 3100 Genetic Analyser.The plasmid 'pCS2', containing the human β-catenin sequence with alanine substitutions at S33, S37, T41 and S45, was kindly provided from Dr. Kemler (Freiburg) and the plasmid designated 'pBatem' containing an N-terminal truncated version of β-catenin missing all phosphorylation sites was kindly received from Dr. Hülsken (Berlin). The plasmid 'βCATa' that was used in some experiments as control was originally constructed as a vector for the production of an anti-sense RNA for β-Catenin by introducing the β-Catenin coding region in an inverse orientation. However, this plasmid was shown to be unable to prevent Wnt-signaling by knocking-out endogeneous β-Catenin mRNA and we therefore used it for normalisation in cotransfection experiments since it has almost the same size as the expression plasmid used in these experiments.Expression plasmids with point mutations were constructed by the introduction of these mutations by site-directed mutagenesis  according to the instructions of the manufacturer. The plasmid 'βCAT_TOTsense', containing the complete coding region for β-catenin under the control of a CMV-promoter was used as a template. The following primer pairs were used to generate the mutant β-catenin plasmids: 'H36P for' (5'-CTT ACC TGG ACT CTG GAA TCC CTT CTG GTG CCA-3') and 'H36P rev' (5'-GAT TCC AGA GTC CAG GTA AGA CTG TTG CTG-3'); 'P44A for' (5'-TCT GGT GGC ACC ACC ACA GCT GCT TCC CTG AGT-3') and 'P44A rev' (5'-ACC TTA GGT AAG ACC ACG GTG GTG GTG TCG-3'); 'S45F for' (5'-GTG CCA CCA CCA CAG CTC CTT TCC TGA GTG GCA-3') and 'S45F rev' (5'-AGG AGC TGT GGT GGT GGC ACC AGA ATG GAT-3'); 'S45P for' (5'-GTG CCA CCA CCA CAG CTC CTC CCC TGA GTG GCA-3') and 'S45P rev' (5'-AGG AGC TGT GGT GGT GGC ACC AGA ATG GAT-3').The mutant β-catenin constructs were verified by restriction enzyme analysis and sequencing using the primer 'Mut_Control 1' (5'-TGC CCT CAT CTA GCG TCT CA-3').2. One day after seeding, the medium was removed and fresh medium was added.Cells from the line HuH7 received a DMEM medium ) supplemented with 10% fetal calf serum , 2 mmol/L glutamine and antibiotics. The cells were cultured at 37°C and 90% humidity, in an atmosphere containing 5% COHindIII site and an EcoRV site (HIII/EVpT81) controlling a reduced herpes simplex thymidine kinase promoter has already been described [The reporter gene with the GS-5'region located between a escribed . This se6 cells per well in 6-well plates in 1.5 ml medium. Twenty four hours after the start of the culture, transfection was performed using the cationic lipid formulation Unifectin-56  according to the manufacturer's instruction. Cells in 6-well plates were transfected with 0.5 μg DNA per well. Forty eight hours after transfection cells from 6-well plates were washed twice with Hank's solution , lysed in 100 μl of lysis buffer  and collected with a rubber policeman. Luciferase measurement was performed as described [For transfection HuH7 cells were cultivated at 0.25 × 10escribed  using a GS: glutamine synthetase, HCC: hepatocellular carcinoma, TBS: Tris buffered saline, POD: peroxidase, LEF-1/TCF: lymphoid enhancer factor-1/T-cell factor, Wnt: wingless-type MMTV integration site family member, GSSEr-BP: glutamine synthetase silencer element from the rat-binding protein.The author(s) declare that they have no competing interests.


We examined the differential expression of β-catenin and its related factors among different types of colorectal cancer in order to determine any relationship with gross tumour morphology. Immunohistochemical staining of β-catenin, E-cadherin and MMP-7 was performed on 51 tumours, including 26 polypoid tumours and 25 ulcerative tumours. Protein truncation tests and single-strand conformational polymorphism for mutation of the adenomatous polyposis coli tumour suppressor gene, as well as single-strand conformational polymorphism for the mutation of β-catenin exon 3 were also done. Nuclear expression of β-catenin was observed in 18 out of 25 (72%) cases of ulcerative colorectal cancer and seven out of 26 (26.9%) cases of polypoid colorectal cancer. A significant relationship of nuclear β-catenin expression with ulcerative colorectal cancer was found (P<0.001). However, this finding was independent of adenomatous polyposis coli tumour suppressor gene mutation and E-cadherin expression. Together with previous data, we propose that different combinations of genetic alterations may underlie different morphological types of colorectal cancer. These findings should be taken into consideration whenever developing a new genetic diagnosis or therapy for colorectal cancer.Although most colorectal cancer develops based on the adenoma–adenocarcinoma sequence, morphologically, colorectal cancer is not a homogeneous disease entity. Generally, there are two distinct morphological types: polypoid and ulcerative colorectal tumours. Previous studies have demonstrated that K-British Journal of Cancer (2002) 86, 1124–1129. DOI: 10.1038/sj/bjc/6600214www.bjcancer.comCancer Research UK© 2002  Colorectal carcinogenesis involves a multistep progression of genetic mutations. Based on the adenoma–carcinoma sequence, much research has focused on mutation detection; sequential genetic alterations have been illustrated as a linear process . AlthougFigure 1el lumen . Ulceratel lumen . These dpolyposis coli (APC) tumour suppressor gene mutation is found in the earliest stage, before proceeding to K-ras mutation as well as to other tumour suppressor genes, such as p53 and DCC. Paralleling increases in adenoma size and grade of dysplasia ensue during colorectal tumour progression. However, nothing has been described on whether different genetic alterations occurred between these two distinct types of colorectal cancer during multistep carcinogenesis. Although data from synchronous adenoma and carcinoma analyses emphasised that the accumulation of genetic alterations is more important than their order  expression, one of the downstream targets of β-catenin, was also analysed in relation to β-catenin expression.We collected 51 primary colorectal carcinoma tissue samples from sporadic colorectal cancer patients who underwent colectomies at Chang Gung Memorial Hospital (CGMH). All samples were collected immediately after resection and stored in a −80°C freezer. Normal mucosa samples were removed at the same time from sites about 10 cm from each tumour. Whole tumour specimens were then prepared for routine histopathological examination. Formalin-fixed, paraffin-embedded colorectal carcinoma tissue samples were preserved in the tissue archives of the Pathology Department at CGMH.Detailed morphological descriptions, histopathological data and clinical data were obtained for each case from the Cancer Registry of the Department of Colorectal Surgery at CGMH.Tumours with exophytic cauliflower-like appearances with or without a very shallow ulcer only, and with a height exceeding half their diameter, were classified as polypoid . TumoursStandard immunohistochemical detection with minor modifications was performed on sections from the archival, paraffin-embedded tissue to detect E-cadherin, β-catenin and matrilysin proteins.Five-micron sections mounted onto slides were deparaffinised and rehydrated in graded alcohols and distilled water. Endogenous peroxidase activity was inhibited by incubation with 3% hydrogen peroxide in methanol for 20 min. Antigen retrieval was done by microwaving at high power for two cycles of 5 min each, with a 10-min break between cycles in citrate buffer at pH 6.0. Non-specific binding of secondary antibodies was blocked by incubation in 10% normal rabbit serum.Incubations with primary antibodies were done at 37°C for 120 min for anti E-cadherin  at 100× dilution, 60 min at 37°C for anti-β-catenin (Transduction Laboratories) at 200× dilution and 1 : 300 for MMP-7 . After three washes with phosphate buffered saline (PBS), the slides were incubated with biotinylated antimouse immunoglobulin and stained using the Ultra Tech Detection System Kit . 3,3′ Diaminobenzidine (DAB) was used as the chromogen. The slides were counterstained with hematoxylin and dehydrated in graded alcohols, air-dried and mounted using a resin-based mounting medium (Immunotech) under coverslips.Slides were independently examined by two experienced observers (JM Chiang and KF Ng) who were blind to the clinicopathological data of the tumour and to the initial results of the other observer. In areas of well-preserved tissue, the staining intensity of the cell membrane or cytoplasm was evaluated using the staining of adjacent non-involved normal mucosa as the internal control for each section. Membrane expression of E-cadherin or β-catenin was considered preserved when staining of cancer cells was as strong as that of normal glands, and the proportion of positive cancer cells in each section was more than 90%. If there was identifiable positive staining in less than 10% of the cancer cells, the tumours were recorded as having a loss of E-cadherin or β-catenin membrane expression. Nuclear staining of positive cells was defined as an intense brown colour in the nucleus .Genomic DNA from each tumour sample and corresponding normal mucosa was extracted. SSCP analysis of β-catenin exon 3 was performed using the following primer pair: exon 3, 5′-GATTTGATGGAGTTGGACATGG-3′ and 5′-TGTTCTTGAGTGAAGGACTGAG-3′. Samples were amplified through 35 cycles on a thermocycler (Perkin-Elmer) at 95°C denaturation for 50 s, 57°C annealing for 30 s and 72°C extension for 10 min. Polymerase chain reaction (PCR) was performed in a volume of 25 μl with 20 ng of genomic DNA in a PCR buffer containing 1.5 mFor the investigation of the APC gene, exon 15 mutations were performed using the protein truncation test (PTT) on genomic DNA as described previously . Exons 1U-test, the Pearson's correlation coefficient and the chi-square test. A two-sided P<0.05 was determined as statistically significant.Comparative data were analysed using the Mann–Whitney We collected 51 colorectal carcinoma samples, including 25 ulcerative tumours and 26 polypoid tumours. The clinicopathological parameters, including age, gender, tumour stage, tumour differentiation and tumour size were comparable between the two groups, except for right colon predominance in the polypoid CRC group and significantly deeper invasion depth in the ulcerative CRC group (Table 1P=0.001) was also observed between the ulcerative  and polypoid (seven out of 26) groups of CRC  and 22 out of the 51 (43%) cases, respectively. Forty-nine per cent (25 out of 51) of cases showed nuclear β-catenin expression and always showed a reciprocal loss of membrane staining except for two preserved cases (Table 3The frequency of APC mutations was 39 out of 51 (76%) cases. Among the 39 mutations found, 34 were detected in exon 15 truncated proteins  tumours. This striking diversity in the gross appearance of CRC may reflect the underlying chaotic genetic instability. In our study, we investigated purely polypoid and purely ulcerative tumours. The implications of our findings are further discussed below.ras codon 12 mutation is selectively related to polypoidal growth of CRC (Contradictory to previous studies showing that K-in vitro studies using cell lines have proven that mutations in the APC tumour suppressor gene occurs in most colorectal cancer and leads to the activation of β-catenin (in vivo. In our study, we showed that nuclear β-catenin expression was independent of APC tumour suppressor gene mutation, as was reported previously (Although many Although a significant relationship exists between ulcerative CRC and nuclear β-catenin expression, we did not find a significant increase in the extent of nuclear β-catenin expression related to the depth of invasion . These fmyc, cyclin D and gastrin reportedly relate to tumour proliferation, while others are related to tumour invasion. MMP-7 is regulated by β-catenin expression (in vivo than in vitro (Precisely how nuclear β-catenin expression confers ulcerative or endophytic growth to CRC remains poorly understood. Further analyses of several downstream factors of the nuclear β-catenin/TCF complex, including c-MYC , gastrinFinally, the finding that nuclear β-catenin expression is closely related to ulcerative growth of CRC further supports a previous study that showed analogies between embryonic gastrulation and β-catenin expression . Strong,In summary, we observed that the different combinations of genetic alterations may selectively underlie different types of CRC. Nuclear β-catenin expression is related to the ulcerative growth patterns of CRC. Although the precise mechanism remains poorly understood, its expression is independent of APC mutation. These observations further highlight the heterogenous nature of CRC, which should be kept in mind when developing a new gene therapy or a new genetic diagnosis for CRC.


Valproic acid (VPA), a commonly used mood stabilizer that promotes neuronal differentiation, regulates multiple signaling pathways involving extracellular signal-regulated kinase (ERK) and glycogen synthase kinase3β (GSK3β). However, the mechanism by which VPA promotes differentiation is not understood.Cip/WAF1 pathway. These effects, however, are not mediated by the pathway involving the epidermal growth factor receptor (EGFR) but via the pathway which stabilizes Ras through β-catenin signaling. Stimulation of differentiation and inhibition of proliferation in NPCs by VPA occur independently and the β-catenin-Ras-ERK-p21Cip/WAF1 pathway is involved in both processes. The independent regulation of differentiation and proliferation in NPCs by VPA was also demonstrated in vivo in the cerebral cortex of developing rat embryos.We report here that 1 mM VPA simultaneously induces differentiation and reduces proliferation of basic fibroblast growth factor (bFGF)-treated embryonic day 14 (E14) rat cerebral cortex neural progenitor cells (NPCs). The effects of VPA on the regulation of differentiation and inhibition of proliferation occur via the ERK-p21We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and inhibition of proliferation in the cerebral cortex of developing rat embryos. VPA als gliomas . VPA is  gliomas ,9. VPA h gliomas ,10,11. V gliomas ,13 inhibCip/WAF1 [VPA has been reported to regulate the differentiation and proliferation of various cells, including mesenchymal and hematopoietic stem cells, neuroblastoma cells, primary neurons, and neural progenitor cells (NPCs) ,14-17. VCip/WAF1 ,6, whichCip/WAF1 . HoweverCip/WAF1 pathway did not occur via the common pathway involving epidermal growth factor receptor (EGFR), an upstream component of the ERK pathway, as indicated by significant reduction in the level of EGFR in the presence of VPA. The level of Ras protein, another upstream component of the ERK pathway, was significantly increased by VPA treatment. This observation led us to conclude that VPA-induced ERK pathway activation occurs via an increase in the stability of Ras, mediated by Wnt/β-catenin signaling [Cip//WAF1 pathway is involved in generating the mutually exclusive phenotypes of differentiation and proliferation in NPCs and in brain tissue of the cerebral cortex of developing embryos.We report here that 1 mM VPA induces differentiation and inhibits proliferation of NPCs by overcoming the effect of basic fibroblast growth factor (bFGF), a factor which inhibits the differentiation of NPCs ,20. VPA-ignaling ,22. We aBasic fibroblast growth factor (bFGF) is necessary for the maintenance of multipotency in neural progenitor cells  and is iCip/WAF1 is an important indicator of ERK pathway-induced inhibition of proliferation [Cip/WAF1 levels in NPCs grown in the presence of bFGF  most often mediates activation of the ERK pathway by extracellular stimuli . AlthougCip/WAF1 pathway and subsequent stimulation of differentiation and inhibition of proliferation induced by VPA in NPCs, we measured the effect of overexpression of β-catenin. Overexpression of β-catenin increased ERK activity and Ras protein level and induced expression of p21Cip/WAF1 and Tuj1  and Tuj1 in the cerebral cortex of the developing rat embryo were distinct and non-overlapping , an alternative transcription target of β-catenin [We have shown that activation of the ERK-p21in liver , EGFR wa-catenin , in NPCs-catenin ,39. We oCip/WAF1 pathway, and subsequent effects on differentiation and inhibition of proliferation in NPCs, was seen in the reduction of the effects of VPA, including ERK activation and induction of p21Cip/WAF1 and Tuj1, following siRNA-mediated β-catenin knockdown. The β-catenin-mediated activation of the ERK-p21Cip/WAF1 pathway following VPA treatment may be attributed to upregulation of Ras, suggested by the increase in the level of Ras seen after β-catenin overexpression. The VPA-induced increase in the level of Ras may be due to the stabilization of β-catenin as a result of inhibition of GSK3?. Increases in Ras following modulation of the Wnt/β-catenin signaling pathway have been demonstrated in various cell types, including primary hepatocytes, and β-catenin has been identified as an important mediator of that process [Cip/WAF1. These results indicate that mutually exclusive patterns of differentiation and proliferation during neuronal differentiation and development may be regulated via the common Ras-ERK-p21Cip/WAF1 pathway involving β-catenin.VPA directly inhibits GSK3β resulting in activation of the β-catenin signaling pathway ,40,41 an process . Regulat process -44; howeCip/WAF1 or siRNA-mediated β-catenin knockdown in several different cases. These results indicate the possibility that VPA-induced differentiation and inhibition of proliferation occur in part via different routes, including, e.g., the pathway affected by inhibition of HDAC [However, the effects of VPA, particularly its effect on proliferation, were modest or only partially inhibited by increases in p21 of HDAC . Althoug of HDAC  and/or iCip/WAF1-pathway mediated by β-catenin. We propose that this mechanism of VPA action may contribute to an explanation of its anti-tumor and neuroprotective effects, as well as elucidate its role in the independent regulation of differentiation and proliferation in the cerebral cortex of developing rat embryos. Regulation of Ras stability in neuronal differentiation and inhibition of proliferation may indicate a new role of the protooncoprotein Ras in the differentiation of stem cells and in development.Our results suggest that VPA induces neuronal differentiation and inhibition of proliferation of cortical NPCs at least partly via the Ras-ERK-p212 at 37°C. NPCs were plated at 4 – 5 × 105 cells per dish in a 60-mm culture dish and grown to 40% confluence, cells were treated with 0.5 – 10 mM (most often 1 mM) VPA for 48 h. To observe the effect of inhibition of MEK, some cultures were co-treated with 20 μM PD98059 (2'-amino-3'-methoxyflavone)  in addition to VPA. To test the effects of overexpression or knockdown, vectors or siRNAs were transfected into NPCs using Lipofectamine TM 2000 (Invitrogen). Transfections were performed in N2 medium without antibiotics, followed by growth in N2 medium containing 50 IU/ml penicillin and 50 μg/ml streptomycin, prior to treatment with VPA. Cultures were photographed using the ECLIPS TE2000-U Fluorescent microscope  equipped with a digital CCD camera .Sprague-Dawley (SD) rats were purchased from KOATECH . All animal procedures were approved by the Institutional Review Board of Severance Hospital, Yonsei University College of Medicine. NPCs were isolated from the cerebral cortex of E14 SD rats as described previously . The isoBeginning on embryonic day 13.5 (E13.5) of gestation, rats were intravenously injected with 200 mg/kg VPA  or phosphate-buffered saline (PBS) twice at 24 h intervals, and provided with water containing 15 g/l VPA for 48 h. On day15.5 embryos were collected for subsequent immunohistochemical analyses.Cip/WAF1, anti-EGFR , anti-p-Raf-1 (Ser-338), anti-pan-Ras , anti-Tuj1 , or anti-β-actin  antibody followed by horseradish peroxidase-conjugated secondary anti-rabbit, anti-rat , or anti-mouse IgG . Protein bands were visualized using enhanced chemiluminescence  and the LAS-3000 imaging system .Cells were rinsed with ice-cold PBS and lysed in 1× Laemmli buffer . Cell lysates were heated at 100°C for 10 min and separated by electrophoresis through 8 – 12% SDS-polyacrylamide gels. Following electrophoretic transfer of proteins to nitrocellulose, membranes were blocked in 5% nonfat dry milk in PBS containing 0.1% (v/v) Tween 20 for 30 min at 25°C followed by incubation with anti-p-ERK, anti-p-MEK, anti-p-GSK3β (Ser-9) , anti-β-catenin , anti-p214 cells/well. When cultures reached 30% confluence VPA was added to the medium. Where indicated, the cells had been transfected with 100 nM p21Cip/WAF1 siRNA or β-catenin siRNA for 12 h before VPA treatment. For the proliferation assay, cultures were incubated with 25 μM BrdU (Sigma-Aldrich) for 2.5 h before fixation. Cells were fixed in 70% ethanol for 30 min, washed three times with PBS, and permeabilized by incubation in 0.1% Triton X-100 in PBS for 30 min. To measure BrdU incorporation, cells were incubated in 2 M HCl for 30 min and washed three times with PBS. The cells were incubated in blocking solution  for 30 min followed by incubation with anti-Tuj1, anti-BrdU , anti-Flag (Sigma-Aldrich), or anti-p21Cip/WAF1 in blocking solution at 4°C overnight. Cells were washed three times in PBS and incubated with Alexa Fluor 488- or Alexa Fluor 555-conjugated IgG secondary antibody  at room temperature for 1 h. Nuclei were counterstained by incubation in 1 μg/ml DAPI  for 10 min followed by exhaustive washing in distilled water. Coverslips were mounted in GelMount . Fluorescent labeling was observed using a Radiance 2100 multi-photon imaging system (Bio-Rad Laboratories) and LSM510META  at excitation wavelengths of 488 nm (Alexa Fluor 488), 543 nm (Alexa Fluor 555), or 405 nm (DAPI). Approximately 300–400 cells were counted in each of three independent experiments to quantify relative p21Cip/WAF1-, Tuj1-, or BrdU- positive cells. For histological analyses, embryos were fixed in 10% neutral buffered formalin for 48 h, dehydrated by serial immersion in alcohols, cleared in xylene, and embedded in paraffin. Four-micron sections were cut using a RM2245 microtome . Immunohistochemical analysis was performed using Alexa Fluor 555- and 488-conjugated IgG secondary antibodies. Antigen retrieval was performed using citrate buffer, pH 6.0. All incubations were carried out in humidified chambers in the dark. Immunofluorescent labeling of tissue sections was performed using the staining procedure described above.For immunocytochemistry, NPCs were plated on coverslips coated with poly-L-ornithine and fibronectin in 24-well plates at a density of 2 – 2.5 × 10Cip/WAF1 (GenBank accession number NM_080782) and β-catenin (GenBank accession number NM_053357) mRNAs were designed using the template design tool . The p21Cip/WAF1 target sequences are 5'-AGACCAGCCUAACAGATTTTT-3' (431–459) and 5'-GAACGGTGGAACTTTGACTTT-3' (136–154). β-catenin target sequences are 5'-AAGGCTTTTCCCAGTCCTTCA-3' (203–223) and 5'-AAGATGATGGTGTGCCAAGTG-3' (1303–1323). β-catenin and p21Cip/WAF1siRNAs were synthesized by Samchully Pharm Co., LTD .Flag-β-catenin-pcDNA3.0 was obtained from Dr. Eric R. Fearon of the University of Michigan School of Medicine. Small interfering RNAs (siRNAs) targeting rat p21bFGF: basic fibroblast growth factor; BMP4: bone morphogenetic protein 4; CP: cortical plate; ERK: extracellular signal-regulated kinase; GSK3β: glycogen synthase kinase3β; HDAC: histone deacetylase; IMZ: intermediate zone; NPC: neural progenitor cells; Tuj1: class III β-tubulin; PCNA: proliferating cell nuclear antigen; SVZ: subventricular zone; siRNA: small interfering RNA; VPA: valproic acid, 2-propyl-pentanoic acid.GAJ, JYY, BSM, DHY, and HYK carried out the experiments. KYC coordinated and wrote the manuscript. SHL, VB, and EA provided reagents and techniques and approved the manuscript.NPCs isolated from E14 embryos retain the capacity to form neurospheres and to differentiate into primary neurons, oligodendrocytes, and astrocytes. NPCs were isolated as described in Methods. (A) Neurospheres were established as previously described [escribed  and mainClick here for fileEffects of different concentrations of VPA on morphological differentiation and proliferation of NPCs. NSCs were isolated from rat cerebral cortex at embryonic day 14 (E14) and cultured in N2 medium supplemented with 10 ng/ml bFGF as described in Methods. At 40% confluence, the cultures were treated with 0, 0.5, 1, 2, 3, or 10 mM VPA and incubated for an additional 48 h. Micrographs were taken using a Nikon ECLIPSE TE2000-U fluorescence microscope at 200× magnification.Click here for fileEffects of MEK inhibitor PD98059 on the morphological differentiation and proliferation of NPCs. NPCs were grown in medium containing 10 ng/ml bFGF. Medium was then replaced with fresh medium containing 10 ng/ml bFGF, 20 μM PD98059, and/or 1 mM VPA, as indicated, and cultures were incubated for an additional 48 h. Cultures were photographed and relative numbers of cells were estimated as described in Figure Click here for fileEffects of bFGF and VPA on proliferation of NPCs. This experiment is similar to that shown in Figure Click here for fileCip/WAF1 siRNA on VPA-induced inhibition of proliferation of NPCs grown in the presence of bFGFEffects of p21. This figure shows the quantitative data for Figure Click here for fileEffects of ß-catenin siRNA on VPA-induced proliferation of NPCs grown in the presence of bFGF. This  figure  shows  the  quantitative  data  for  Figure  Click here for fileMeasurements of NPC transfection efficiencies.      NPCs were  grown  in N2 medium  containing bFGF  (10 ng/ml),  and transfected  with  0,  0.5,  1,  2,  or  3  µg  pEGFP-C1  (Clontech)  as  described  in Methods.    Percentages  of  GFP-positive  cells  were  estimated  24  h  after transfection.    Error  bars  indicate  the  standard  deviation  of  three  independent experiments.  Click here for file