The Wnt/β-catenin signaling pathway plays essential roles in cell proliferation and differentiation, and deregulated β-catenin protein levels lead to many types of human cancers. On activation by Wnt, the Wnt co-receptor LDL receptor related protein 6 (LRP6) is phosphorylated at multiple conserved intracellular PPPSPXS motifs by glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1), resulting in recruitment of the scaffolding protein Axin to LRP6. As a result, β-catenin phosphorylation by GSK3 is inhibited and β-catenin protein is stabilized. However, how LRP6 phosphorylation and the ensuing LRP6-Axin interaction lead to the inhibition of β-catenin phosphorylation by GSK3 is not fully understood. In this study, we reconstituted Axin-dependent β-catenin phosphorylation by GSK3 and CK1 in vitro using recombinant proteins, and found that the phosphorylated PPPSPXS peptides directly inhibit β-catenin phosphorylation by GSK3 in a sequence and phosphorylation-dependent manner. This inhibitory effect of phosphorylated PPPSPXS motifs is direct and specific for GSK3 phosphorylation of β-catenin at Ser33/Ser37/Thr41 but not for CK1 phosphorylation of β-catenin at Ser45, and is independent of Axin function. We also show that a phosphorylated PPPSPXS peptide is able to activate Wnt/β-catenin signaling and to induce axis duplication in Xenopus embryos, presumably by inhibition of GSK3 in vivo. Based on these observations, we propose a working model that Axin recruitment to the phosphorylated LRP6 places GSK3 in the vicinity of multiple phosphorylated PPPSPXS motifs, which directly inhibit GSK3 phosphorylation of β-catenin. This model provides a possible mechanism to account, in part, for inhibition of β-catenin phosphorylation by Wnt-activated LRP6. Drosophila, dorsal-ventral axis formation in Xenopus, and homeostasis of the mammalian gastrointestinal tract adenomatous polyposis coli (APC), phosphorylates β-catenin at Thr41, Ser37, and Ser33 β-TrCP E3 ubiquitin ligase complex. The SCFβ-TrCP ubiquitin ligase poly-ubiquitinates β-catenin, leading to β-catenin degradation via the proteosome pathway The Wnt/β-catenin signal transduction pathway plays central roles in many aspects of cell proliferation and differentiation, such as segment polarity determination in Wnt activates the β-catenin pathway via two distinct classes of receptors on the cell surface: one is a member of the Frizzled family of seven-transmembrane receptors, and the other is a single transmembrane receptor referred to as LDL receptor related protein 6 (LRP6), or its relative LRP5. Wnt may induce a Frizzled-LRP6 coreceptor complex et al. reported that the LRP6 intracellular domain directly inhibits GSK3 phosphorylation of β-catenin in a PPPSPXS-dependent manner et al. suggested that LRP6 specifically inhibits GSK3 phosphorylation of β-catenin but not of other substrates How PPPSPXS phosphorylation and its recruitment of Axin result in inhibition of β-catenin phosphorylation remains a critical question. To address this issue we established an in vitro β-catenin phosphorylation system using recombinant Axin, GSK3 and CK1. We found that each of the multiple phosphorylated PPPSPXS peptides inhibits the phosphorylation of β-catenin at Ser33/Ser37/Thr41 by GSK3 in a sequence and phosphorylation-dependent manner. This inhibition is specific for GSK3, as these phospho-peptides do not affect β-catenin Ser45 phosphorylation by CK1, and occurs regardless of the presence or absence of Axin. We also found that a phosphorylated PPPSPXS peptide is able to activate Wnt/β-catenin signaling and to induce axis duplication in Xenopus embryos, presumably via inhibition of GSK3 in vivo. These results suggest a potential mechanism to account, in part, for the inhibition GSK3 phosphorylation of β-catenin by the activated LRP6. While this manuscript was in previous review processes, Cselenyi E. coli or baculovirus-infected insect cells, and purified these proteins to over 90% homogeneity by affinity chromatography  or an antibody specific for Ser33/Ser37/Thr41-phosphorylation (by GSK3).To study how β-catenin phosphorylation is regulated by upstream components of the Wnt pathway, we reconstituted an in vitro kinase assay for β-catenin amino-terminal phosphorylation using purified proteins. We overexpressed recombinant β-catenin, Axin, CK1β, and GSK3β proteins in either tography . We incuWhen CK1 was present in the kinase reaction, β-catenin was strongly phosphorylated at Ser45 . When CKA single phosphorylated PPPSPXS motif is sufficient to activate β-catenin signaling in vivo When the phosphorylated PPPSPXS peptides were added to the in vitro β-catenin phosphorylation assay, the Phos-A, Phos-C, as well as Phos-E peptide each inhibited, interestingly, β-catenin phosphorylation at Ser33/Ser37/Thr41 by GSK3 . The PhoSince Axin is a scaffolding protein critical for GSK3 phosphorylation of β-catenin and binds to the phosphorylated PPPSPXS motif As demonstrated in S motif CK1 phosphorylates β-catenin at Ser45, which is the priming site for β-catenin Ser33/Ser37/Thr41 phosphorylation by GSK3 E. coli, and purified mGS-CTD by GST column affinity chromatography  and the Tau protein tography . We recot Ser641 . When CKt Ser641 , recapitt Ser641 . GSK3 alt Ser641 , as detet Ser641 . In addit Ser641 . These rAs the dually phosphorylated Phos-A peptide inhibits GSK3 phosphorylation of β-catenin in vitro, we examined whether Phos-A could inhibit GSK3 and activate β-catenin signaling in vivo. It has been well established that inhibition of GSK3, such as via a dominant-negative GSK3 mutant, activates Wnt/β-catenin signaling and induces axis duplication in Xenopus embryos Although it is known that the activated Wnt/Frizzled/LRP6 receptor complex results in inhibition of β-catenin degradation, the underlying molecular mechanism has remained unclear. One critical initiating event is Wnt-induced LRP6 phosphorylation at multiple PPPSPXS motifs CPPSPXS motif  specificity of phospho-LRP6 inhibition of GSK3 phosphorylation. Cselenyi et al reported that the LRP6 intracellular domain specifically inhibits GSK3 phosphorylation of β-catenin, but not of Tau Cselenyi One potential mechanism for the observed inhibition of GSK3 by the phosphorylated PPPSPXS peptide is substrate competition, given that the PPPSPXS motif is a substrate for GSK3 In summary, we propose a working model to link LRP6 activation and the inhibition of β-catenin phosphorylation. When activated by Wnt, PPPSPXS motifs in LRP6 intracellular domain are phosphorylated by GSK3 and CK1, and they act together to recruit Axin-GSK3 into the Wnt receptor complex. When Axin is bound to one of the phosphorylated PPPSPXS motifs, other phosphorylated PPPSPXS motifs in the vicinity of the Axin-GSK3 complex directly inhibit GSK3 phosphorylation of β-catenin . SeveralWe note that our above model  does notWhile this manuscript was being revised for publication Piao et al reported that a dually phosphorylated PPPSPXS peptide  inhibits GSK3 phosphorylation of both β-catenin and Axin in vitro, and a LRP6 intracellular fragment containing a single PPPSPXS motif inhibits GS phosphorylation when overexpressed in the cytoplasm The pGEX4T1 vector expressing wild-type β-catenin (GST-tagged) was described The GST-tagged β-catenin and GST-tagged mGS-CTD were expressed by transforming expression plasmids into BL21 (DE3) cells, grown in LB Broth, and induced by 0.5 mM IPTG. Bacterial cells were then lysed by three cycles of freeze-thaw. The cell lysate was centrifuged at 14,000 rpm for 30 min, and the supernatant was incubated with glutathione agarose resin (Sigma). The bound proteins were washed by buffer and eluted by 7 mM glutathione. Baculoviruses for Flag-tagged CK1, His-tagged GSK3, and MBP-tagged Axin were amplified in the Sf9 insect cells (Invitrogen), and then used to infect the High-Five insect cells (Invitrogen), which were harvested two days after infection and lysed by freeze-thaw cycles. The M2 anti-Flag agarose resin (Sigma) was used to purify Flag-tagged CK1. The amylose resin (New England Biolab) was used to purify MBP-tagged Axin. The Ni-NTA agarose resin was used to purify His-tagged GSK3. The purified Tau protein was purchased from Calbiochem. Flag-Axin, Flag-AxinΔDIX, and Flag-Axin(351-701) were overexpressed by transfecting HEK293T cells with the respective expression plasmids, and purified using M2 anti-Flag agarose resin (Sigma) from cell extracts lysed in buffer with 0.5% NP40.SPXS), and were further phosphorylated by CK1 at the second serine site (PPPSPXS) at 37°C for 3 hours in vitro.The HA, 14-3-3BP, Phos-A, A-mut, Phos-C, Phos-D, and Phos-E peptides were synthesized by Tufts University Core Facility, purified by reverse phase HPLC, and their identities were confirmed by mass spectrometry. The synthesized peptides were dissolved in Tris buffer (pH 8.0) to 500 µM, and dialyzed by the 3,500 Dalton Molecular-Weight-Cut-Off Slide-A-Lyzer MINI dialysis units overnight to remove chemicals used in peptide synthesis. Recovery of peptides after dialysis was estimated to be 30% for all peptides using dot-blotting analysis via respective antibodies Transfections were done in HEK293T cells in 6-well plates using Fugene-6 (Roche). 48 hours after transfection, cells were lysed in a buffer containing 10 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 10 mM NaF, and 0.5% NP-40 with a cocktail of protease inhibitors. The cell lysates were centrifuged at 14,000 rpm for 10 minutes, and the supernatant of the cell lysates were taken for further studies.2, 2 mM DTT, and 10 mM ATP (pH 7.0), in a total volume of 15 µl at 37°C for 3 hours. For Axin-dependent phosphorylation of β-catenin, 0.43 µM of GSK3, 0.54 µM of CK1α, 0.21 µM of Axin, and 0.73 µM of β-catenin were used in each assay. For Axin-independent phosphorylation of β-catenin, the condition was the same except that 2.2 µM of GSK3 (and no Axin) were used in each assay. For phospho-peptide (or control peptide) inhibition experiments, 1 µl of the dialyzed peptide  or four-fold serially diluted peptide  was used in each assay. For GS phosphorylation, 0.50 µM of CK2 proteins, 0.43 µM of GSK3, and 0.8 µM of GST-mGS-CTD were used in each reaction. For Tau phosphorylation, 0.43 µM of GSK3 and 0.8 µM of Tau protein were used in each reaction. Each in vitro assay was repeated three or more times.The phosphorylation assays were performed in buffer containing: 25 mM Hepes pH 7.5, 20 mM β-glycerophosphate, 25 mM MgClThe supernatant of cell lysates or kinase reaction products were examined by SDS-PAGE, and analyzed by Western blotting using Immobilon-P membrane (Millipore). The membranes were incubated in blocking buffer (5% nonfat dry milk in TBS buffer with 0.1% Tween-20) for 1 hour at room temperature, and then were incubated with primary antibodies diluted in 1% BSA in the TBS/Tween-20 buffer for 1 hour, followed by incubation with horseradish peroxide-conjugated secondary antibodies diluted at 1∶10,000 in 1% BSA in the TBS/Tween-20 buffer for 30 minutes. Protein detection was performed using the ECL system (Amersham Pharmacia). The following antibodies were used : anti-phospho-Ser33/Ser37/Thr41 β-catenin (1∶1000) from Cell Signaling (9561S); anti-phospho-Ser45 β-catenin (1∶1000) from Cell Signaling (9564S); anti-β-catenin (1∶1000) from BD Biosciences (610153); anti-phospho-Ser641 glycogen synthase (1∶1000) from Cell Signaling (3891S); and anti-Tau (1∶1000) from Cell Signaling (4019). The PHF1 anti-phosphor-Tau antibody was a kind gift from Peter Davies (Albert Einstein School of Medicine).Embryo manipulations and RT-PCR were described previously


The entorhinal cortex is perhaps the area of the brain in which neurofibrillary tangles and amyloid plaques are first detectable in old age with or without mild cognitive impairment, and very particularly in Alzheimer's disease. It plays a key role in memory formation, retrieval, and extinction, as part of circuits that include the hippocampus, the amygdaloid nucleus, and several regions of the neocortex, in particular of the prefrontal cortex. Lesions or biochemical impairments of the entorhinal cortex hinder extinction. Microinfusion experiments have shown that glutamate NMDA receptors, calcium and calmodulin-dependent protein kinase II, and protein synthesis in the entorhinal cortex are involved in and required for extinction. Aging also hinders extinction; it is possible that its effect may be in part mediated by the entorhinal cortex. Since extinction is perceived as the waning of a CR, it might be takenfor the expression of forgetting . Howeverβ-adrenergic antagonist, propranolol . Retrieval oftentriggers a reconsolidation of learned responses [Some recent evidence, however, raises the alternative possibility thatextinction may involve the actual erasure of a memory trace. Extinction can bemade more intense by increasing the time that rats are exposed to the absenceof a footshock US in an inhibitory avoidance conditioning paradigm. In these conditions,spontaneous recovery may not be (readily) seen, and the reinstallment of theoriginal avoidance response requires again gene expression and proteinsynthesis in the hippocampus , as it wlol (see ), and thlol . Indeed, we forget the face of people we sawjust once or twice and then never again, unless they were highly arousing oremotionally important , 33. Mem5. IndeedSeveral fMRI studies show an activation of prefrontal areas, notably theventromedial prefrontal cortex (vmPFC), together with reduced blood flow in thebasolateral amygdala (BLA) –42 and/oSeveral early studies using localized brain lesion or stimulationtechniques –64 and oThe molecular basis of inhibitory avoidance  andotheIt is widely agreed that aging is accompanied by a cognitive declineboth in laboratory animals and in humans. Behavioral and molecular aspects ofthis decline have been studied extensively in the last two decades (see ). RecentPerhaps the region of the brain which ages more rapidly is theentorhinal cortex. Normal aging has been known for many years to beaccompanied by a reduction of neuron and synapse counts in many regions of thecerebral cortex, particularly the entorhinal cortex and then the hippocampus.The earliest occurrence of prototypical lesions in Alzheimer's disease isusually considered to be in the entorhinal cortex (,JellingThe entorhinal cortex plays a key role in cognition. It contributes to,and processes, information that the rest of the cortex, particularly theprefrontal areas, sends to it in order to be relayed to the hippocampus andamygdala, as part of the acquisition, retrieval, or extinction of many forms oflearning. In addition, the entorhinal cortex also processes information generatedby the hippocampus and sends it to the neocortex, and interconnects thehippocampus with its main regulatory nucleus complex, the amygdala. Thus theentorhinal cortex is crucially involved in all aspects of learning. Its role inextinction has been best studied in aversive tasks. The entorhinal cortex onone hand and extinction on the other suffer severe losses with aging. Thechanges are more marked in humans with mild cognitive impairment, and muchworse in Alzheimer's disease, in which the entorhinal losses are diagnostic atearly stages. The impairment of extinction seen in old age may be related inpart to entorhinal cortex damage.


Its role in urothelial cancer (TCC) is unclear, since typical activating mutations are not found. We therefore determined the activity of a β-catenin/TCF-dependent promoter in proliferating normal uroepithelial cells and seven TCC cell lines, using a hepatoma line with oncogenic β-catenin as a control. Neither normal urothelial cells nor TCC lines exhibited activity under normal growth conditions. In normal cells and 5/7 TCC lines, even transfection of activated β-catenin did not restore promoter activity, suggesting repression of β-catenin/TCF activity. TCF mRNAs and total β-catenin protein levels did not differ qualitatively between inducible and noninducible cell lines, but E-cadherin expression was lacking or low in inducible TCC lines. In these, cotransfection of E-cadherin diminished activation of the TCF-dependent promoter by β-catenin. Our results make constitutive WNT/β-catenin signalling in TCC appear unlikely, thereby explaining the lack of reported mutations. However, decreased E-cadherin expression occurring in many TCC, often as a consequence of promoter hypermethylation, may confer inappropriate responsiveness to WNT factors.Constitutive activation of WNT signalling through  In the absence of activating WNT signals, β-catenin is phosphorylated by a multiprotein complex containing GSK3β, the tumour suppressors APC and axin/conductin. Phosphorylated β-catenin is recognised by a ubiquitin–ligase complex with β-TrCP as the crucial component, ubiquitinylated and degraded by the proteasome. Binding of an activating WNT factor to the Frizzled receptor leads to the inactivation of GSK3β via activation of Dishevelled and GBP/FRAT and thus to stabilisation of β-catenin, which accumulates in the cytoplasm, translocates into the nucleus and heterodimerises with TCF/LEF transcription factors to activate target genes containing specific TCF binding sites in their promoters. Constitutive activation of WNT/β-catenin signalling in cancer is caused by a variety of mutations in various components of the pathway, leading either to inactivation of APC, axin/conductin or β-TrCP or to oncogenic activation of β-catenin. In addition, recent evidence suggests that the activity of the pathway may also be influenced by further factors, notably by the status of the E-cadherin cell adhesion protein  is unclear. WNT/β-catenin signalling is implicated in urothelial development. Expression of Wnt2b in mesenchymal renal cells induces ureter branching in the murine foetus  or high frequency (CDH1) in advanced TCC and could conceivably lead to increased β-catenin/TCF activity and proliferation in TCC . Transitional cell bladder carcinoma lines harbour genetic changes and gene expression patterns typical of advanced TCC and provide a well-characterised and established experimental system to study properties of this tumour. Normal uroepithelial cells can be maintained in primary cultures, where they proliferate spontaneously or stimulated by external growth factors such as EGF. Furthermore, we analysed the expression of β-catenin, APC and E-cadherin at the protein level and expression of TCFs at the mRNA level. The effect of E-cadherin was investigated in more detail. Overall, our data suggest that proliferation of TCC does not depend on activity of the WNT/β-catenin pathway, which instead appears to be generally repressed. E-cadherin may act as a buffer for β-catenin activity in urothelial cells and its loss in TCC may not only affect cell adhesion, but also sensitivity towards WNT factors.To circumvent the vagaries associated with mutation detection in multiple candidate genes and to determine the significance of reported changes in μg/ml penicillin/streptomycin as described , 5 ng/ml epidermal growth factor (EGF) and 30 ng/ml cholera toxin. After the first passage, they were used for experiments as described , supplemented with 15% fetal calf serum and 100 Photinus pyralis luciferase gene, were purchased from Upstate Biotechnology . The Renilla reniformis luciferase reporter plasmid, pTKRL, used as internal control for transfection efficiencies, and the negative control vector pGL3 were purchased from Promega, Mannheim, Germany. pLINEluc, constructed by insertion of bps −193 to +661 from the active LINE-1 element L1.2B into pGL3  as recommended by the manufacturer. Each experiment was repeated with at least five independent passages or cultures.Cells were grown in six-well plates to 30% confluence. Transient transfection was carried out using FuGene/DMEM  at a 1 : 25 dilution. Per well, 1 2 tissue culture flasks to 80% confluence and lysed for 1 h on ice in modified RIPA-buffer . Protein amounts were quantified by the Bradford method. For SDS–polyacrylamide gel electrophoresis , 10 μg of protein were used and subsequently transferred to an Immobilon-P membrane . After blocking in 10% nonfat milk powder in PBS overnight at 4°C, membranes were incubated for 1 h at room temperature with primary antibodies at the following dilutions: anti-β-catenin  at 1 : 1500, anti-E-cadherin  at 1 : 1000, anti-APC  at 1 : 500 and anti-α-tubulin  at 1 : 5000. Incubation with HRP-conjugated rabbit anti-mouse secondary antibody (Santa Cruz Biotechnology) at 1 : 5000 was carried out at room temperature for 1 h, followed by luminescence detection with the ECL-Kit .Cells were grown in 75 cmμl volume containing 1 × PCR-buffer , 150 μM of each nucleotide, 10 pmol of each primer, 1 U of Taq polymerase (Biometra) with 2% (CTNNB1), 3% (TCF1), 2% (TCF4) and 2.5% (hAES) formamide added. Each PCR cycle consisted of 30 s denaturing at 95°C, 30 s at the annealing temperature and 1 min at 72°C. Each initial denaturation was performed for 5 min at 95°C and each final extension for 7 min. A total of 25 cycles were performed for CTNNB1 at 57°C annealing temperature, 34 cycles at 59°C for TCF1, 35 cycles at 58°C for LEF1, 31 cycles at 59°C for TCF3, 29 cycles at 56°C for TCF4, and for hAES, 36 cycles at 59°C. Primer pairs were selected according to CTNNB1, TCF4 and TCF1, LEF1, TCF3 and hAES. GAPDH, with primers added to the mixture for the last 19 cycles, served as an internal control. Polymerase chain reaction products were separated by agarose gel electrophoresis (2%) and visualised by ethidium bromide staining.Total mRNA was isolated from cultures grown to 80% confluence, using the RNeasy® Midi Kit . After quantification, mRNA was transcribed into first-strand cDNA using AMV-RT . Polymerase chain reactions were carried out in a total 20 μg DNA from each cell line was bisulphite-treated with the CpGenome DNA modification kit . Aliquots from the reaction mixture were then used in two separate PCR amplifications with primer pairs from the CDH1 and the APC gene promoter regions specific for converted, that is unmethylated, or unconverted, that is methylated, DNA using the conditions specified in μl containing 150 μM of each dNTP, 1.5 mM MgCl2, 15 pmol of each primer, and 1.25 U of HotStar Taq polymerase . Following initial denaturation at 94°C for 15 min, 35 cycles of 30 s at 95°C, 30 s at Tm, and 45 s at 72°C were performed. All reactions included a final elongation step at 72°C for 10 min.High molecular weight genomic DNA from cell lines was isolated using the blood and cell culture DNA kit . MS-PCR was performed essentially as described by APC and CDH1 hypermethylation status, which were newly determined. By MS-PCR, all APC alleles were hypermethylated in HT1376, and one allele of CDH1 was hypermethylated in SW1710.Some important properties of the TCC lines used in this study are compiled in β-catenin pathway was determined in proliferating NUEC and seven TCC lines under optimal growth conditions. The hepatoma cell line HepG2 harbouring an activating β-catenin mutation was used as a positive control. The cells were transfected with either pTopFlash or pFopFlash plasmids. These plasmids are identical but for mutations in the TCF/β-catenin binding sites in the promoter driving luciferase expression. The ratio of luciferase expression from pTopFlash to pFopFlash is an established indicator of Wnt/β-catenin pathway activity. Differences in transfection efficiencies were corrected by cotransfection of a Renilla luciferase plasmid. Plasmids without a promoter (pGL3) or with a cell-type-independent retrotransposon promoter (pLINEluc) were included in each experiment as quality controls.First, endogenous activity of the WNT/β-catenin mutation, HepG2 displayed significant endogenous signalling activity (12.3±3.3). In contrast, the ratios found in normal urothelial cells (0.96±0.09) and in the TCC lines (range: 0.64±0.20–1.33±1.3) indicate lack of β-catenin/TCF signalling.Figure 1Aβ-catenin was able to induce promoter activity from pTopFlash in TCC and NUEC in cotransfection experiments. vs without β-catenin. Surprisingly, in normal urothelial cells and four of seven TCC-lines, the ratio did not differ significantly from 1 with values ranging from 0.2±0.1 in normal urothelial cells to 2.4±2.1 in VMCub1, indicating that expression of activated β-catenin was not sufficient to increase transcription from the β-catenin/TCF-dependent promoter. In contrast, significant induction was observed in the TCC lines SW1710 (12.7±2.3) and 5637 (58.7±23.8). A slight, but not statistically significant induction was observed in BFTC905 (4.9±2.0). The high basal activity in HepG2 was not further increased by transfected β-catenin, as expected. Cotransfection of β-catenin did not significantly alter the activity of pFopFlash .In addition to methylation analysis of the β-catenin signalling pathway is an important step in the development of many human cancers. However, none of the typical mutations in components of this pathway have been reported in TCC. Few studies have appeared addressing this issue explicitly, which is likely due to publication bias against negative results (β-catenin signalling pathway are rare or absent in TCC. Moreover, the data suggest that this pathway is inactive in and not required for proliferation of TCC cells. Since the same result was obtained with proliferating NUEC, the lack of basal activity in TCC cells is likely an extension of the state in the normal tissue. Moreover, even in normal cells, the pathway could not be activated by oncogenically activated β-catenin. Thus, the WNT/β-catenin signalling pathway is not only inactive, but turned off in urothelial cells, likely because it is not required for proliferation and perhaps to avoid inappropriate target gene activation. More speculatively, it is interesting to consider the downregulation of the WNT/β-catenin pathway in urothelial cells and carcinomas in the light of current hypotheses on the function of the pathway in maintaining stem cell properties (Constitutive activity of the WNT/operties . Accordiβ-catenin target genes such as CCND1 and MYC is required for the proliferation of uroepithelial cells, too. Proliferation of NUEC is stimulated by growth factors such as EGF and, in an autocrine fashion, HB-EGF (CCND1 and MYC in an alternative fashion to WNT/β-catenin signalling. Almost all advanced TCC display defects in either RB1 or CDKN2A, which obliterate the requirement for cyclin D1 (cf. CCND1 itself is amplified (MYC is found in almost all TCC, and is correlated with increased gene copy numbers (MYC or CCND1, required for the proliferation of TCC cells, is likely achieved by mechanisms other than activation of WNT/β-catenin signalling.Nevertheless, the activation of typical WNT/n D1 cf. . Further numbers . Thus, uβ-catenin pathway has become defective. We did not observe significant differences between these two cell lines and the others in expression of overall β-catenin, hLEF1, hTCF3 and hTCF4. However, both cell lines displayed decreased expression of E-cadherin. Indeed, restoration of E-cadherin expression caused repression of TCF/β-catenin-induced transcriptional activity (β-catenin signalling, since VMCub2 cells also lacked the protein, but did not respond to β-catenin transfection. Interestingly, both inducible TCC lines, but not VMCub2, displayed slightly increased mRNA expression of hTCF1 and hAES. If hTCF1 acts as a feedback repressor of β-catenin/TCF4 signalling (β-catenin. However, the hAES homologue Grg5 is known to act as a de-repressor of TCF-mediated transcription (β-catenin/TCF signalling in SW1710 and 5637. The potential roles of hTCF1 and hAES are more difficult to address experimentally than that of E-cadherin, since the differences in hTCF1 and hAES expression between the cell lines were only quantitative.This interpretation implies that the inducibility of TCF-dependent gene expression found in the TCC lines 5637 and SW1710 is a deviation from the normal state in the urothelium and that at least one of the mechanisms ensuring inactivity of the WNT/activity . On a noβ-catenin signalling in urothelial cells is in line with recent data suggesting this as a function of E-cadherin additional to or coordinate with mediating cell adhesion (β-catenin pathway may play a certain role in a subset of TCC, likely those with loss of E-cadherin expression. This subset of tumours may show increased sensitivity towards WNT factors present in the tissue. Indeed, changes in WNT7B expression have been reported in some TCC (The finding that E-cadherin modulates WNT/CDH1 and APC genes has been reported in TCC tissues. Hypermethylation of CDH1 was found to be associated with loss of protein expression, although not all cases with loss of protein also displayed hypermethylation (APC hypermethylation, reported at frequencies up to 30% (APC alleles, but neither basal activity nor inducibility of the WNT/β-catenin pathway were observed. In fact, APC protein expression could be detected in HT1376. While this suggests that APC hypermethylation does not implicate activation of WNT/β-catenin signalling, more extensive studies are required on this issue.Finally, hypermethylation of the


CTNNB1) stabilizing mutation in pHPT tumors.Hyperparathyroidism (HPT) is a common endocrine disorder with incompletely understood etiology, characterized by enlarged hyperactive parathyroid glands and increased serum concentrations of parathyroid hormone and ionized calcium. We have recently reported activation of the Wnt signaling pathway by accumulation of β-catenin in all analyzed parathyroid tumors from patients with primary HPT (pHPT) and in hyperplastic parathyroid glands from patients with uremia secondary to HPT (sHPT). Mechanisms that may account for this activation have not been identified, except for a few cases of β-catenin  mouse model. WNT3 ligand and the internally truncated LRP5 receptor strongly activated transcription, and the internally truncated LRP5 receptor was insensitive to inhibition by DKK1.Reverse transcription PCR and Western blot analysis showed expression of an aberrantly spliced internally truncated WNT coreceptor low-density lipoprotein receptor–related protein 5 (LRP5) in 32 out of 37 pHPT tumors (86%) and 20 out of 20 sHPT tumors (100%). Stabilizing mutation of The internally truncated LRP5 receptor is strongly implicated in deregulated activation of the WNT/β-catenin signaling pathway in hyperparathyroid tumors, and presents a potential target for therapeutic intervention. Gunnar Westin and colleagues report the expression of an aberrantly spliced LRP5 receptor in primary and spontaneous parathyroid tumors and implicate it in the deregulated activation of the Wnt/β-catenin signaling pathway. The parathyroid glands—four rice-sized glands in the neck—maintain a normal calcium balance in the body, to maintain strong bones and essential cellular functions. The glands release parathyroid hormone as a response to a decrease in blood calcium level. By stimulating calcium release from bone and its absorption in the gut, parathyroid hormone restores the blood calcium level. However, 100,000 new individuals in the US develop hyperparathyroidism (HPT) annually, characterized by enlarged, overactive parathyroid glands and high blood levels of calcium. Primary HPT (pHPT) is usually caused by a benign tumor (a non-life-threatening growth) in one of the parathyroid glands. Secondary HPT (sHPT) occurs in response to calcium regulatory disturbances, linked to vitamin D deficiency, and more or less invariably develops in patients with uremic kidney disease.HPT is usually treated by surgical removal of the enlarged parathyroid glands, which is done with great efficiency. However, ideally, doctors would like to know what drives the overgrowth of the parathyroid glands to be able to develop drugs for treatment or disease prophylaxis. Researchers recently reported that the cells in enlarged parathyroid glands from patients with HPT contain high amounts of β-catenin. This protein is part of the Wnt signaling pathway, which has been found to be disrupted in many tumor entities in other organs. In the absence of Wnt proteins, a group of proteins called the β-catenin destruction complex marks β-catenin so that it is rapidly destroyed. When Wnt proteins bind to a cell-surface receptor called Frizzled and a coreceptor called LRP5, the destruction complex is inhibited and β-catenin accumulates. This accumulation induces the production of other proteins  that stimulate cell growth and division. The accumulation of β-catenin in the enlarged parathyroid glands of patients with HPT could, therefore, significantly contribute to the overgrowth of their glands—but what causes β-catenin accumulation? In this study, the researchers have investigated this question to try to identify a target for drugs to treat HPT.The researchers looked for genetic changes (mutations) in β-catenin that stabilize the protein and measured the expression of LRP5 in abnormal parathyroid gland tissue from 37 patients with pHPT and 20 with uremia and sHPT. All the samples contained high levels of β-catenin, but only four contained a β-catenin–stabilizing mutation. All the sHPT samples and 32 pHPT samples (but none of the samples containing the β-catenin stabilizing mutation) expressed a mutated LRP5, with the central region deleted. To investigate the functional consequences of this internally deleted LRP5 protein, the researchers used a technique called RNA interference to block its expression in a human parathyroid tumor cell line. They found that expression of the mutated, short LRP5 is required for accumulation of β-catenin, expression of c-Myc, and continued growth of the cell line in test tubes and in animals.The accumulation of β-catenin in all the enlarged parathyroid glands examined so far strongly implicates abnormal Wnt/β-catenin signaling in the development of pHPT and sHPT. These new findings identify which part of the signaling pathway is altered. The expression data and functional data together suggest that an internally deleted LRP5 coreceptor is often responsible for the accumulation of β-catenin. The functional data also show that expression of shortened LRP5 is necessary for the abnormal growth of parathyroid tumor cells. Exactly how the internally deleted coreceptor activates β-catenin signaling in parathyroid gland cells, or why a shorter-than-normal LRP5 is made, are not yet known. However, because these findings indicate that internally deleted LRP5 has a fundamental role in activating Wnt signaling in HPT, drugs that inactivate this aberrant protein but leave the normal protein unscathed might provide a nonsurgical treatment for this common hormone disorder.http://dx.doi.org/10.1371/journal.pmed.0040328.Please access these Web sites via the online version of this summary at hyperparathyroidism, primary hyperparathyroidism, and secondary hyperparathyroidim (in English and Spanish)edlinePlus has encyclopedia pages on hyperparathyroidism, which includes links to organizations that help people with hyperparathyroidismInformation is available for patients from the US National Institute of Diabetes and Digestive and Kidney Diseases on Wnt signaling pathway and on β-catenin Wikipedia maintains pages on  Secondary hyperparathyroidism (sHPT) develops in patients with uremia because of phosphate retention, hypocalcemia, and reduced 1,25-dihydroxyvitamin Dcalcemia . Parathyeoplasia ,7, was fell line . Overexpell line . In a smpromoter . AugmentCTNNB1 or inactivating mutations in APC is strongly implicated in the cause of approximately 10% and 80% of colorectal cancers, respectively [Dysregulated Wnt signaling with accumulation of β-catenin in the cytoplasm/nucleus plays an important role in a variety of human cancers. The stability of β-catenin is regulated by Wnt ligands through a “destruction complex” consisting of APC/Axin/GSK-3β/Ck1/Dvl and other factors. In the absence of Wnt ligand, free cytoplasmic β-catenin is rapidly degraded by the proteasome after phosphorylation of its amino terminus at residues serine 33, serine 37, threonine 41, and serine 45 –15. Wnt ectively ,14. A muectively . In thisn = 37) and hyperplastic glands (n = 20) from patients with pHPT and sHPT, respectively, were acquired from patients diagnosed and operated on in the clinical routine. Each patient contributed with one tumor. All 57 tumors displayed aberrant accumulation of β-catenin (unpublished data), of which 14 parathyroid adenomas and all 20 hyperplastic parathyroid glands were described previously [n = 6) was obtained from glands inadvertently removed in conjunction with thyroid surgery where autotransplantation was not required or as normal parathyroid gland biopsies in patients subjected to parathyroidectomy. All tissues were intraoperatively snap-frozen, and cryosections were used in the analyses. Written informed consent and approval of local ethics committee was obtained.Parathyroid adenomas (http://www.ncbi.nlm.nih.gov/Genbank). A total of 1%–2% of the primary PCR product was used for nested PCR. Primers used were the following: forward primer, 5′-CTTCACCAGCAGAGCCGCCATCCACAG-3′; nested forward, 5′-GGATCTCCCTCGAGACCAATAACAACG-3′; and reverse, 5′-CCGGGATCATCC GACTGATG-3′. The PCR amplifications were performed with cDNA, 25 pmol of each primer, 0.2 mM dNTPs, 1× PCR buffer, 1.5 mM MgCl2, and 0.25 U Platinum Taq DNA polymerase (Invitrogen). The PCR conditions were: denaturation at 95 °C for 60 s, followed by 40 cycles of denaturation for 20 s, annealing at 58 °C for 20 s and extension at 72 °C for 90 s, and a final extension at 72 °C for 7 min. An annealing temperature of 61 °C and 40 cycles were used for nested amplification. Two LRP5wt and six randomly chosen truncated LRP5 cDNA fragments from parathyroid tumors, as well as from sHPT-1 cells, were cloned into pCRII-TOPO (Invitrogen), and sequenced on ABI 373A using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems). All fragments encoded an open reading frame, and the seven truncated fragments contained the same in-frame deletion (Δ666–809). Normal tissue cDNA or RNA were purchased from BD Biosciences Clontech and Ambion.Total RNA was extracted with TriZol Reagent  according to the manufacturer's instructions and the RNA was subsequently treated with RQ1 DNase I (Promega) and proteinase K. Alternatively, DNA-free RNA was prepared using the Nucleospin RNA II kit (Macherey-Nagel). Successful DNase treatments were established by PCR analysis of all RNA preparations. Reverse transcription of total DNA-free RNA was performed with random hexamer primers using the First-Strand cDNA Synthesis kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. cDNA was amplified by primary or nested PCR using mRNA-specific primers spanning positions 1992–2932 of LRP5  were distributed onto 35-mm dishes in DMEM/10% fetal bovine serum and subsequently harvested at the indicated time points. The number of viable cells were determined by using the NucleoCounter (ChemoMetec). Cells were collected at 84 h after siRNA transfection and incubated with FITC-labeled annexin V to assess phosphatidylserine externalization as a marker for apoptosis.Cells .cDNA was prepared as described above. The following mRNA-specific PCR primers and labeled probes (5′FAM-sequence–3′TAMRA) were used for quantitative real-time RT-PCR analysis. For LRP5wt: forward: 5′-CCTGAAGACCATCAGCCGCG-3′; reverse, 5′-CCCGCTCCTGACCCAGCATG-3′; and probe, 5′-TCCCACCAAGGGCTACATCTACTG-3′. For LRP5tot: forward, 5′-ATCGACTGTATCCCCGGGGC-3′; reverse, 5′-CACCACGCGCTGGCACACAA-3′; and probe, 5′-CGGACTGTGACGCCATCTGCCTGC-3′. For MYC: forward, 5′-AAGACTCCAGCGCCTTCTCTCCGT-3′; reverse, 5′-TGGGCTGTGAGGAGGTTTGCTGTG-3′; and probe, 5′-AGCGACTCTGAGGAGGAACAAGAA-3′. For 28S rRNA, the Ribosomal RNA Control Reagents (VIC probe) was used (Applied Biosystems). PCR reactions were performed on ABI PRISM 7700 Sequence Detection System or MyiQ Single-Color Real-Time PCR Detection System (Bio-Rad) using the TaqMan PCR core Reagent Kit (Applied Biosystems). Each cDNA sample was analyzed in triplicate. Standard curves for the expressed genes were established by amplifying a purified PCR fragment covering the sites for probes and primers. Messenger RNA-specific PCR primers for WNT1 were as described . Primers5 cells/35-mm dish with jetSI-ENDO according to the manufacturer's recommendations (Poly-Plus-Transfection SAS). The following siRNAs were used: control nonsilencing siRNA (Qiagen Operon), siLRP5Δ666–809; 5′-TAACAACGACCUCACCAUUdTdT-3′ and 5′-AAUGGUGAGGUCGUUGUUAdTdT-3′ , siLRP5wt; 5′-CAACCACAUCUACUGGACAdTdT-3′ and 5′-UUCCAGUAGAUGUGGUUGdTdT-3′ (Thermo Electron), siLRP5tot; stealth 5′-CCUGCAUGGACUGAGGAACGUCAAA-3′ and stealth 5′-UUUGACGUUCCUCAGUCCAUGCAGG-3′, control siLRP5tot; stealth 5′-CCUGGUAGUCAAGGAUGCACCGAAA-3′ and stealth 5′-UUUCGGUGCAUCCUUGACUACCAGG-3′ (Invitrogen). A very high transfection efficiency  was obtained for sHPT-1 cells by this protocol [sHPT-1 parathyroid tumor cells  were traprotocol  luciferaprotocol  for 24 hprotocol  mouse moMYC promoter DNA, containing TCF-4 binding site 2 [Chromatin immunoprecipitation (ChIP) of transfected cells was performed using a protocol from Upstate, but with immunoprecipitation conditions as described . The antg site 2 , was PCR6 cells for 24 h. The animals were monitored every day and humanely killed after 8–9 wk. The animal experiments were approved by the Uppsala University board of animal experimentation and were performed according to the United Kingdom Coordinating Committee on Cancer Research guidelines for the welfare of animals in experimental neoplasia [Female Fox Chase severe combined immunodeficiency (SCID) mice (2- to 3-wk-old) were used (Taconic). The mice were anesthetized with isoflurane  during the manipulations. One flank or both flanks of each animal were injected subcutaneously  with sHPT-1 cells together (1:1) with BD Matrigel Matrix (BD Biosciences Clontech) after transfection of 10eoplasia .t test was used for all statistical analyses. Values are presented as arithmetrical mean ± standard error of the mean. A p-value below 0.05 was considered significant.Unpaired n = 57) which all displayed aberrant cytoplasmic/nuclear accumulation of β-catenin  was detected in 32 out of 37 pHPT tumors (86%) and 20 out of 20 sHPT tumors (100%) by using RT-PCR primers located in exons 9 and 13 of LRP5 A. Of the09 was deThe tumor-associated shorter transcript LRP5Δ666–809 contained an in-frame deletion of 142 amino acids (Δ666–809), encompassing the third YWTD β-propeller domain ,29 betweTo investigate the functional consequences of LRP5Δ666–809 expression, if any, specific and control siRNAs were efficiently transfected [MYC [MYC mRNA level was significantly reduced in sHPT-1 parathyroid cells transfected with siLRP5Δ666–809 and siLRP5tot, but not with siLRP5wt, control siRNA, and control siLRP5tot , and transcription was most prominently stimulated (80-fold) in the presence of WNT3 ligand, where it was 4-fold higher than for LRP5wt A. In addd tumors B.Several amino acid residues in the deleted part of LRP5 (Δ666–809) were shown to be required for inhibition of β-catenin activity by the WNT antagonist DKK1 . TherefoThe in vivo growth properties of sHPT-1 cells were evaluated in a xenograft SCID mouse model. Significant tumor growth inhibition was observed in transplants of cells when pretransfected with siLRP5Δ666–809 or siLRP5tot, but not with siLRP5wt or control siRNA A and 6B.CTNNB1-stabilizing mutations in a few cases (3 out of 20) of pHPT tumors, while no mutation was found in uremic secondary HPT tumors, and inactivating truncations of APC were not seen [CTNNB1-stabilizing mutations in exon 3 [CTNNB1 mutation frequency in pHPT tumors [CTNNB1-stabilizing mutations has been found so far, suggesting an overall mutation frequency of approximately 6% . This frequency is slightly lower than that observed for colorectal cancers, in which approximately 10% of the tumors harbored missense mutations or interstitial deletions of exon 3 [β-catenin accumulation has been observed by us in all so far analyzed parathyroid tumors of primary origin and hyperplastic parathyroid glands of HPT secondary to uremia  and in hyperplastic parathyroid glands of HPT secondary to uremia (100%). Of all 57 analyzed parathyroid tumors with accumulated β-catenin, 52 expressed the internally truncated LRP5 receptor without LRP5 missense mutations, which cause a high-bone-mass phenotype, are located before the first epidermal growth factor–like repeat in the amino terminal part of the receptor, not including the third YWTD β-propeller domain, which is absent in the truncated LRP5 receptor. Several mutations of LRP5 known to cause osteoporosis-pseudoglioma syndrome is located within amino acids 666–809, highlighting a functional role for this part of the protein [All reported  protein . Osteopo protein . ClearlyMDM2 oncogene in breast cancer [LRP5 gene in the analyzed tumors that potentially could explain the aberrant splicing. Mutations in other areas of the LRP5 gene affecting splicing specificity, accuracy, or efficiency, or perhaps more likely interference of trans-acting factors of the splicing machinery, are other possibilities. Alternative splicing may be affected in a splicing factor concentration-dependent manner [RNA splicing defects in cancer are common, but seem rarely to be caused by somatic mutations in splice sites and regulatory elements. Alternative splicing may potentially present as a diagnostic marker ,39. Intet cancer . We fount manner , and thiThe results support a fundamental role of the aberrantly spliced internally truncated LRP5 receptor in activating WNT/β-catenin signaling in parathyroid tumor cells. LRP5 receptors could be attractive targets for the development of antitumor drugs that specifically inactivate the truncated receptor and leave the normal protein unaffected.


Immortalised human ovarian surface epithelium and ovarian cancer cell cells (OVCAR-3) expressed β-catenin, APC, GSK3β and Lef-1. Nuclear staining of β-catenin and Lef-1 were demonstrated only in OVCAR-3 cells. There were significant increases of β-catenin and GSK3β, while APC was reduced in ovarian cancer compared to the normal ovary. Beta-catenin and Lef-1 were coimmunoprecipitated in ovarian tumours, but not in the normal ovary. Nuclear localisation of β-catenin or Lef-1 could not be demonstrated in the normal ovary or in the ovarian tumours. The absence of nuclear localisation of β-catenin could be due to an increased binding to the cadherin–α-catenin cell adhesion complex. In fact, we have earlier reported an increased expression of E-cadherin in ovarian adenocarcinomas. In summary, this study demonstrates an increase in the expression of components of the Wingless/Wnt pathway in malignant ovarian tumours. The increase suggests a role for this signalling pathway in cell transformation and in tumour progression. However, it remains to be demonstrated whether it is an increased participation of β-catenin in transcriptional regulation, or in the stabilisation of cellular integrity, or both, that is the crucial event in ovarian tumorigenesis.Beta-catenin is involved in both cell–cell adhesion and in transcriptional regulation by the Wingless/Wnt signalling pathway. Alterations of components of this pathway have been suggested to play a central role in tumorigenesis. The present study investigated, by immunohistochemistry and immunoblotting, the protein expression and localisation of  The formation of this complex depends on the GSK3β phosphorylation status. In the normal cell, degradation of β-catenin through the ubiquitin pathway is facilitated by phosphorylation  either in the cytoplasm or in the nucleus. This is followed by transcriptional activation of target genes, that is, c-myc, E-cadherin and cyclin D1  cadherin, but not epithelial (E) cadherin, is expressed in the normal ovarian surface epithelium (OSE) . Tissues were immediately washed in ice-cold 0.9% NaCl, snap frozen in liquid nitrogen and stored at −70°C until analysis. Ovarian tumours were classified according to surgical staging. All samples were examined by two independent and experienced gynaecological pathologists for histological diagnosis and grade -sample buffer and heated before loading on a 1D SDS-polyacrylamide gel (8% Tris-Glycine) or NuPage gel (10%) . Proteins were transferred onto a polyvinyldifluoride (PVDF) membrane  using a blotting system (NOVEX). The membrane was then incubated with antibodies against β-catenin , GSK3β,  , α-catenin  APC-COOH terminus or APC-NH2-terminus , Tcf-Lef  and Lef-1  or Lef-1 antiserum (1 : 10000) . The optical density (OD) of each band was measured. For quantification, a reference sample, same on each blot, was included. The standard was set to 100%. The signal from each band was then correlated to the standard and this relative number was used in the statistical analysis.β-catenin (1 : 100), Lef-1 (1 : 100), Lef-1 antiserum (1 : 100), GSK3β (1 : 50), APC-COOH and APC-NH2-terminus (1 : 100) and incubated overnight. Bound antibodies were visualised by biotinylated secondary horse anti-mouse or goat anti-rabbit antibodies  and Streptavidin-FITC . In control sections, which showed negligible signals, the first antibody was replaced by normal mouse IgG  or 5% NFM  and Catch-and-Release  according to the protocols provided by each manufacturer, with modifications. Briefly, a soluble lysate of an ovarian serous adenocarcinoma, poorly differentiated, stage III, was prepared as described above with the exception of the homogenisation buffers that were provided with the kits. The lysates  were incubated with the primary antibody overnight at 4°C on a rotating platform. The eluted fractions were concentrated by ethanol precipitation  and vacuum-dried. The pellets were dissolved in an SDS-sample buffer. Separation by SDS–PAGE and immunodetection was performed as described above.post hoc Wilcoxon rank-sum test and used in the analysis of immunoblotting data obtained by the densitometric scanning of membranes. A P-value less than 0.05 was considered to be significant. Values are given as mean±s.e.m.The nonparametric Kruskal–Wallis test for comparing groups was followed by the β-catenin at the cell borders  in the analyses and anti-APC NH2-terminus was found to be the most reliable for both IHC and immunoblots. This antibody recognizes full-length APC and truncated APC  and GSK3β (P<0.001) in the group of ovarian adenocarcinomas (AC) as compared to the group of normal ovaries (N) and benign adenomas/borderline tumours (BB)  from the same patient was included. The normal colon sample, normal ovaries, benign adenomas and borderline-type tumours exhibited higher expression of a band at the expected molecular weight (312 kDa) compared to the adenocarcinomas, OVCAR-3 cells and CRC  and one undifferentiated adenocarcinoma (stage III) .Immunoprecipitation was performed in order to detect a physical interaction between β-catenin immunoprecipitates, expression of GSK3β or APC was not detected (data not shown). However, silver staining of the gel for the immunoprecipitates after transfer revealed a band with a molecular weight of approximately 300 kDa in the adenocarcinoma, suggesting APC as an interacting partner with β-catenin in this tumour, in addition to Lef-1 (data not shown). In addition, β-catenin was also immunoprecipitated with α-catenin . The β-catenin band was also present in immunoprecipitates with Tcf/Lef and Axin (lanes 4 and 6).The immunoprecipitation with Lef-1 in the ovarian tumour sample also contained a band for β-catenin, earlier only known as an important protein in cadherin-mediated cell–cell adhesion, also plays a crucial role in Wnt-signalling. In the normal cell, binding of β-catenin to APC, GSK3β and Axin will lead to degradation of β-catenin through the ubiquitin–proteasome pathway. The importance of β-catenin in the Wnt-pathway was originally described in colon and melanoma cells (In recent years, it has become clear that β-catenin, GSK3β, APC and Lef-1. Their localisation to these specific epithelial cells was demonstrated by immunohistochemistry. Others, as well as the results of this study, have revealed that β-catenin is localised at the cell borders and not in the nucleus of normal OSE or OSE lining the inclusion cysts (β-catenin has been described in normal cells of other organs such as the endometrium of the uterus (β-catenin gene are infrequent in ovarian carcinoma and interestingly only described in the endometrioid type of epithelial ovarian tumours (β-catenin gene in ovarian cancers (β-catenin/Lef in normal ovarian tissue. We therefore conclude that a complex of β-catenin/Lef, for transcriptional regulation, is less likely to be formed in normal OSE.Ovarian surface epithelium is commonly believed to be the origin for ovarian adenocarcinomas, which arise in inclusion cysts lined with OSE . In our β and APC in the human ovary is not known. However, as for other epithelial cells, it could be speculated that GSK3β and APC regulate the free pool of β-catenin in normal ovarian cells. Normal APC function seems to be critical for the colorectal mucosal epithelium, since APC mutations are often found in the earliest stages of colorectal tumorigenesis. The APC protein has been demonstrated in several subcellular compartments including the cytoplasm, nucleus and adhesive cadherin–catenin junctions (β-catenin. We have localised GSK3β to the cytoplasm and the cell borders of OSE lining the ovary and inclusion cysts. The expression of both GSK3α and GSK3β were previously described in the normal ovary (The function for GSK3unctions . Other tβ-catenin and GSK3β in the adenocarcinomas as compared to normal ovarian tissue and benign adenomas. Adenomatous polyposis coli expression was decreased or absent in ovarian adenocarcinomas, while Lef-1 was constantly expressed in all the tissues analysed. This indicates a possible role for the Wnt-pathway in ovarian tumour cells. Several members of the Tcf-Lef family are expressed in colorectal cancer cells and malignant mammary epithelium (β-catenin and Lef-1, indicating dysfunction in normal Wnt-signalling, was found in the OVCAR-3 cells but not in ovarian tumour biopsies. Nuclear β-catenin was recently noted in 12 out of 96 serous adenocarcinomas (β-catenin in an ovarian serous adenocarcinoma and in the OVCAR-3 cell line. Constitutive β-catenin/Lef-1-mediated transcriptional activity has been detected in four out of 19 investigated ovarian cancer cell lines (In the present study, we found a significant increase of ll lines .β-catenin/Lef1, relocate β-catenin to the cell border and inhibit the degradation of β-catenin (β-catenin to the plasma membrane. This would then reduce the free pool of cytoplasmic β-catenin and inhibit nuclear translocation, reflected by the absence of nuclear β-catenin in ovarian tumours in this study. In fact, the absence of E-cadherin can lead to an accumulation of free β-catenin and increased transactivation, which can be competed out by transient transfections with E-cadherin (β-catenin proteolytically at the N- and C-terminal, which will reduce α-catenin binding, release actin filaments and reduce cell–cell interaction, indicates that β-catenin has an important role at a late stage of apoptosis. Studies on oral squamous cell carcinoma cells also indicate that cadherin-mediated adhesion promotes anchorage-independent growth and suppresses apoptosis (β-α-catenin complex in ovarian cancer could make the cancer cells less susceptible to apoptosis and thereby favour growth.Beta-catenin has additional functions in the cell since it also binds to the cadherin cell–cell adhesion complex. In fact, both N- and E-cadherin can reduce or block the transcriptional activity of poptosis . A functβ-catenin in the cytoplasm (2-terminus antibody, low or absent levels of full-length APC protein were noted in adenocarcinomas and OVCAR-3 cells even though staining of tumour cells was positive. A lower band that might represent a truncated form of APC was demonstrated in these samples. It has been suggested that the NH2-terminal fragment of APC is sufficient to localise APC to the apical membranes together with GSK3β, Axin, dishevelled and β-catenin forming a high molecular weight complex that catalyses the degradation of β-catenin (In patients linked to familial adenomatous polyposis, truncations of or mutations in the APC gene are believed to enhance the progression of colorectal or gastric tumours, presumably through stabilisation of -catenin . The recβ-catenin complex results in accumulation of cytoplasmic β-catenin and nuclear localisation. Nuclear transcriptional activation by β-catenin/Tcf-Lef of specific targeted genes, that is, c-Myc and cyclin D, which are upregulated in ovarian tumour, could be important steps in tumour initiation and/or progression (Mutational changes of members of the APC/Axin/gression . The posβ-catenin–GSK3β complex. Lef-1/β-catenin precipitates were also found in malignant ovarian cancer, but without concomitant staining in the nucleus of the tumour cells. The proteosomal degradation of β-catenin could still be active or β-catenin might be focused at the cell–cell junctions via the high E-cadherin expression in ovarian tumours, which in turn could favour tumour growth. In contrast to other tumours, the transcriptional activity of Lef-1/β-catenin might play a minor role in ovarian epithelial tumours. Further studies with an in vitro approach need to be conducted to elucidate whether the transcriptional activity of β-catenin–Lef-1 and/or increased cell-junction stability is of importance in ovarian tumorigenesis.In conclusion, this study demonstrates a significant change in the expression of some components in the Wnt/Wingless-signalling pathway in ovarian epithelial tumours. This could indicate dysfunction of the APC–


Pancreatic ductal adenocarcinoma (PDA) is an extremely aggressive malignancy, which carries a dismal prognosis. Activating mutations of the Kras gene are common to the vast majority of human PDA. In addition, recent studies have demonstrated that embryonic signaling pathway such as Hedgehog and Notch are inappropriately upregulated in this disease. The role of another embryonic signaling pathway, namely the canonical Wnt cascade, is still controversial. Here, we use gene array analysis as a platform to demonstrate general activation of the canonical arm of the Wnt pathway in human PDA. Furthermore, we provide evidence for Wnt activation in mouse models of pancreatic cancer. Our results also indicate that Wnt signaling might be activated downstream of Hedgehog signaling, which is an early event in PDA evolution. Wnt inhibition blocked proliferation and induced apoptosis of cultured adenocarcinoma cells, thereby providing evidence to support the development of novel therapeutical strategies for Wnt inhibition in pancreatic adenocarcinoma. Pancreatic ductal adenocarcinoma (PDA) is the fourth leading cause of cancer death in Western societies. The disease usually presents at an advanced stage, and as a result only 10 to 20% of patients are suitable for surgical resection Interactions between embryonic signaling pathways ensure proper organ formation during development. Increasing evidence suggests that these pathways remain active in a subset of cells within adult organs and that deregulation of their activity contributes to the development and progression of certain tumors A large body of evidence supports the notion that uncontrolled activation of the canonical Wnt pathway induces tumor formation in the distal gastrointestinal tract Sustained tumor growth due to deregulation of Wnt signaling independent of mutations that increase pathway activity has been demonstrated in studies on breast and ovarian cancer Here we identify aberrant expression of Wnt signaling components in a large cohort of patients with PDA. Whilst only 13% of PDA demonstrate nuclear localization of ß-CATENIN, 65% demonstrate either loss of membranous expression and/or increased cytoplasmic expression. Similar results were obtained from the analysis of mouse models of pancreatic cancer. Assessment of PDA cell lines revealed activation of Wnt signaling and pathway inhibition showed that cancer cell survival and proliferation depends in part on Wnt activity. Increased levels of Hedgehog signaling constitute one of the earliest changes in PanIN lesions. Our results show that Hedgehog signaling activates ß–CATENIN/Wnt signaling in transgenic mice and untransformed pancreatic duct cells, suggesting that Hedgehog may play a role in upregulating Wnt activity in PDA.A number of recent reports have characterized the role of Wnt signaling during pancreas development and function of the adult organ To obtain more quantitative information about the level of Wnt signaling, we performed expression profiling of Wnt pathway components in PDA with genechip microarray. This analysis demonstrated aberrant expression of numerous components of the Wnt pathway in 12 PDA samples when compared to control tissue  . ExpressKras oncogene have been observed in PanIN lesions, suggesting that deregulation of Ras signaling is a key component during the transformation of epithelial cells G12DPdx-Cre;Kras transgenic mice in which expression of the prevalent G12DKras mutation in pancreatic epithelial cells results in the formation of tumors that resemble human PDA G12DPdx-Cre;Kras and G12D;p53f/+Pdx-Cre;Kras mice by immunohistochemistry. In support of the human data, PanIN lesions in G12DPdx-Cre;Kras and G12D;p53f/+Pdx-Cre;Kras mice displayed changes in ß-catenin localization and increased expression of Tcf4, a mediator of canonical Wnt signaling  , Wnt ligand co–receptors LRP5 and LRP6, and transcription factors TCF3 and TCF4, in 26 human PDA cell lines derived from either primary or metastatic PDA  we measured luciferase expression controlled by concatemers of ‘TCF optimal sites’ (Top) upstream of a minimal thymidine kinase (TK) promoter element IcatLef-1 (dn-Lef-1), resulted in reduction of Top-Flash activity in the 4 PDA cell lines tested , a line with high metastatic potential (L3.6sl), and a line closely resembling a primary tumor phenotype (Panc4.21) were examined s tested . In contIN siRNA . Thus, WIcat and dn-Lef-1. The presence of an IRES sequence driving the expression of eGFP in the Icat- and dn-Lef-1-constructs allowed sorting and comparison of GFP/target gene negative control and GFP/target gene positive cells by flow cytometry. Transfection of the four pancreatic cancer cell lines with either Icat or dn-Lef-1 led to a significant decrease in cell proliferation and a marked increase in apoptosis . In addition, a similar reduction in cell proliferation and increase in apoptosis was seen with dn-Lef-1 transfection, that specifically blocks ß-CATENIN mediated transcription . Expression of the transgenes was confirmed by immunofluorescence  followed by an IRES-eGFP sequence. Target gene expression in pools of transfected cells was verified by Western blot and by flow cytometry using eGFP as a marker (data not shown). Top–Flash reporter assays confirmed an approximately 4–fold increase in Wnt activity in PDC–Lef1–da cells compared to PDCs. However, ectopic activation of Wnt signaling did not induce luciferase activity of the Hedgehog responsive Gli-BS reporter . We have previously reported that forced activation of Hedgehog signaling in Pdx-Cre;CLEG2 mice results in the formation of undifferentiated tumors G12DPdx-Cre;CLEG2;Kras, To test whether Hedgehog activation is sufficient to induce Wnt/ß-catenin signaling Notably, both Hedgehog and Wnt signaling appear to be important for the survival of pancreatic adenocarcinoma cells. This raises the question whether the cooperation between these pathways is restricted to the pancreas or whether similar mechanisms can also be found in other tumors. With the exception of the colon where conflicting results regarding the relationship between Hedgehog and Wnt signaling have been reported earlyPdx1-Cre mice    to generter mice . All stuAxioscop 2 plus microscope .Histological analysis of tissues was performed as described previously 2 for 1 hour and stained with LacZ solution  NP40, and 0.01%(w/v) sodium-deoxycholate in PBS) at room temperature for 24 hours. After staining, tissues were post-fixed with buffered formalin, embedded in paraffin, sectioned, and counterstained with nuclear fast red.Mice were perfused with ice-cold fixative [0.25% glutaraldehyde in phosphate-buffered saline (PBS)]. Pancreas samples were fixed for 90 minutes at 4°C. Tissues were then washed with PBS containing 2 mM MgClTumor cell lines and cell culture protocols were previously described RNA from pancreatic cancer cells and PDCs was prepared using TRIzol reagent . 2 µg of total RNA were treated with DNAse RQ1 (Promega) prior to cDNA synthesis . The oligonucleotide sequences are described in the supplemental information .Anti-β-catenin and anti-GLI1 siRNA sequences were previously described . The GliBrdU incorporation assays were performing using the BD Biosciences BrdU flow kit according to the manufacturer's instructions.Additional methods are provided in Figure S1Immunostaining of β-CATENIN in human normal and cancer tissues. A. In control human pancreas β-CATENIN is localized at the cell membrane in both acinar and duct cells. B, C, D. In three different human PDA samples β-CATENIN is localized predominantly in the cytoplasm (D) and nucleus .(9.42 MB TIF)Click here for additional data file.Figure S2Quantitative PCR for Wnt target genes  in pancreatic samples isolated from three wild type control and three Pdx-Cre;KrasG12D mice. All mice analyzed were 6 months old. Data are presented compared to expression of the household gene GUS.(0.36 MB TIF)Click here for additional data file.Figure S3Inhibition of Wnt signaling induces apoptosis in pancreatic cancer cell lines. A. Relative apoptosis is measured by FACS using an anti-cleaved caspase 3 antibody in cells transfected with a control siRNA  or β-catenin siRNA (black bars). B. Immunostaining for cleaved caspase 3 in cells transfected with a control siRNA or β-catenin siRNA.(1.78 MB TIF)Click here for additional data file.Figure S4The Hedgehog signaling pathway acts upstream of the Wnt signaling pathway in pancreatic cancer cells. A. Activity of the Top-Flash vector in pancreatic adenocarcinoma cells (black bars) is inhibited in response to cyclopamine (gray bars), an inhibitor of the Hedgehog signaling pathway. The first P-values indicate statistical significance in Top-Flash activation (black bars) in comparison to Fop-Flash activity (white bars). The second P-values show significance of reduction in Top-Flash activity upon cyclopamine treatment (gray vs black bars). B. Immunostaining of pancreatic cancer cells grown in control conditions or treated with cyclopamine for 24 hrs. With the exception of BxPC# cells, cyclopamine treatment strongly inhibits TCF4 expression in the cell lines analyzed. Green: anti-TCF4 antibody; blue: DAPI. C. Quantification of the percentage of TCF4 positive nuclei in control cells (white bars) and in cyclopamine-treated cells (black bars). P-values are shown in comparison to the control cells. Error bars are shown as St. Dev.; p-values #, not significant; *p<0.05; **, p<0.01.(1.71 MB TIF)Click here for additional data file.Table S1Upregulation of Wnt receptors, Wnt ligands and β-CATENIN itself as well as downregulation of some inhibitors of Wnt signaling (IDAX and ICAT), together with genes whose transcription is mediated by β-CATENIN, suggests activation of Wnt signaling in PDA.(0.18 MB DOC)Click here for additional data file.Methods S1(0.05 MB DOC)Click here for additional data file.


The E-cadherin–catenin complex plays an important role in establishing and maintaining intercellular connections and morphogenesis and reduced expression of its constituent molecules is associated with invasion and metastasis. In the present study, we examined E-cadherin and α-, β- and γ-catenin levels in tumour tissues obtained by radical prostatectomy in order to investigate the relationship with histopathological tumour invasion. Immunohistochemical findings for 45 prostate cancer specimens demonstrated aberrant expression of each molecule to be associated with dedifferentiation and, in addition, alteration of staining patterns for the three types of catenin was significantly correlated with capsular but not lymphatic or vascular invasion. The data thus suggest that three types of catenin may be useful predictive markers for biological aggressiveness of prostate cancer. © 1999 Cancer Research Campaign


Oral leukoplakia is a precancerous change developed in the oral mucosa, and the mechanism that oral leukoplakia becomes malignant through atypical epithelium is not known. Here we compared the β-catenin expression detected by immunohistochemical staining in the normal oral epithelium and in the oral leukoplakia with or without dysplasia.The normal oral epithelium showed β-catenin expression only in the cell membrane, but not in the nuclei. In the oral leukoplakia without dysplasia, 7 out of 17 samples (41%) showed β-catenin expression in the cell membrane, and 5 samples (29%) showed expression in the nuclei. In the oral leukoplakia with dysplasia, nuclear expression of β-catenin was shown in 11 out of 12 samples (92%). Incidence of nuclear β-catenin expression was significantly different between dysplasia and normal oral epithelium (P < 0.01), and also between oral leukoplakia with dysplasia and those without dysplasia (P < 0.01). Wnt3 expression was detected in the epithelial cell membrane or cytoplasm in oral leukoplakia where nuclear expression of β-catenin was evident, but not in epithelial cells without nuclear expression of β-catenin.The components of canonical Wnt pathway, such as Wnt3, β-catenin, and cyclin D1, were detected, implying that this pathway is potentially involved in the progression of dysplasia in oral leukoplakia. Oral leukoplakia is known as a precancerous change of squamous cell carcinoma developed in oral mucosa -3. LeukoThere are many reports on β-catenin accumulation into a nucleus of a cancer cell in the epithelial malignant tumor, including colorectal cancer -8, and tBecause Wnt family is known to participate in epithelial cell proliferation, we examined in this study immunohistochemical localization of β-catenin, with attention to transcription activation of a target gene by β-catenin, and to evaluate nuclear accumulation β-catenin in relevance with atypical epithelium in a surface layer of oral leukoplakia. Nuclear localization of β-catenin is correlated with cyclin D1 expression in oral leukoplakia, and also Wnt3 expression in neighboring cells, known as a typical member of the Wnt family that activates β-catenin-mediated signaling ,19, suggThe expression patterns of β-catenin in oral mucosa are summarized in Table Localization of β-catenin in the epithelial cell membranes was observed in normal oral epithelium and oral leukoplakia, whereas expression in OSCC was low or totally absent in the cell membrane Fig. . The expWe examined the expression pattern of Wnt3 in oral leukoplakia. Wnt3 was not expressed in the normal oral epithelium (data not shown). Figure We also examined other members of Wnt family, including Wnt1, Wnt5a, and Wnt7a, for immunohistochemical staining. No signal was detectable with antibodies against Wnt1 and Wnt7a in normal and leukoplakia epithelia, whereas Wnt5a signal was ubiquitously detectable in normal oral epithelium, leukoplakia, and in OSCC (data not shown). Thus, these Wnt members are unlikely to be involved in nuclear localization of β-catenin because of the absence of correlation.Cyclin D1 was intensely expressed in the oral leukoplakia where nuclear expression of β-catenin is evident, in contrast to those without nuclear expression of β-catenin Fig. . Cyclin β-Catenin has been identified to be a constituent of cell adhesion apparatus bound to cadherin family , and plaIn contrast to membranous and cytoplasmic expression, nuclear expression of β-catenin is implicated in tumor progression, but the significance of β-catenin localization in the oral epithelial dysplasia and OSCC has not yet been examined in details. The nuclear expression of β-catenin increased during progression of the severity in oral leukoplakia. Furthermore, because localization of β-catenin in normal oral epithelium and dysplasia showed significant difference, suggesting nuclear expression of β-catenin as aberrant condition. In addition, nuclear expression of β-catenin was also observed in 10 out of 15 samples of OSCC, and membranous β-catenin expression was low or totally absent in 7 out of 15 samples of OSCC (data not shown). In various malignant tumors, reduced membranous β-catenin expression was associated with the emergence of invasion or metastasis -24. Our c-Myc and cyclin D1 have been identified as target genes of the Wnt/β-catenin pathway -17. TetsOur studies show the aberrant Wnt signaling pathway in the oral epithelial dysplasia. Wnt3, a typical member of the Wnt family that activates β-catenin mediated signaling ,19, was These lines of evidence suggest that nuclear accumulation of β-catenin plays an important role during malignant transition of oral leukoplakia through dysplasia. In addition to pathological examination of hematoxylin and eosin-stained sections for oral leukoplakia, immunochemical staining for β-catenin is useful for diagnosis of epithelial dysplasia and precancerous changes. Evaluation of other constituents for Wnt signaling, including GSK-3, CDK, Frizzled, and LRP5/6, helps us for determination and prognosis of precancerous change of dysplasia.As for the transcriptional activation of a target gene by β-catenin, elevated expression was detected in a squamous cell carcinoma and epithelial dysplasia of oral leukoplakia. Thus, Wnt/β-catenin pathway is considered to be involved in the progression of dysplasia in oral leukoplakia, as shown by nuclear expression of β-catenin and other components, including Wnt3 and cyclin D1.Formalin-fixed, paraffin-embedded samples of normal oral mucosa (6 examples), of oral squamous cell carcinoma (15 examples), and of oral leukoplakia (29 examples) were used. A fresh tissue of the oral cavity is also obtained at the time of biopsy or at oral surgery during treatment of cancerous disease after obtaining informed consent to the patients, and fixed in formalin before paraffin embedding. A histopathological diagnosis of each sample was performed by pathological staffs in the Department of Pathology in Kawasaki Medical School Hospital. According to the WHO diagnostic criteria for histological typing of cancer and pre-cancer of the oral mucosa [Sections of 4 μm thickness in paraffin embedding tissue were treated with microwave for 10 minutes in 10 mM citrate buffer (pH 6) after de-paraffin processing, followed by washing with PBS twice for 10 minutes. To remove endogenous peroxidase activity, sections were treated in 3% hydrogen peroxide solution for 15 minutes, and subsequently in PBS twice for five minutes. The primary antibodies against β-catenin (Sigma-Aldrich), Wnt3 (Santa Cruz Biotechnology), cyclin D1 (Zymed Laboratories), and c-myc (Santa Cruz Biotechnology) were used at 1/400, 1/100, 1/50, and 1/50 dilution, respectively, in PBS by incubating at room temperature for 60 min. After washing with PBS, the sections were treated with biotinylated secondary antibody for 10 min and then with peroxidase-labeled streptavidin for 10 min , and the color was developed with 3,3'-diaminobenzidine tetrahydrochloride , followed by counter staining with Mayer's hematoxylin. As a negative control, primary antibodies were omitted and the sections were incubated with the secondary antibody alone.For counting cells with nuclear and/or cytoplasm staining, three microscopic fields with 200-fold magnification were randomly chosen and the mean above 10% of the total cell numbers was deduced to be positive.U-test, and Student's t-test.Evaluation of staining was carried out based on whether nuclear or cytoplasmic staining is detectable. Under microscopic field of 200-fold magnification, tissues are positive for β-catenin, if more than 10% of cells show cytoplasmic or nuclear staining. Randomly selected field in the tumor parenchymal region was used for determination of β-catenin positive cells in squamous cell carcinoma. Because signal intensity and distribution were different depending on the places in leukoplakia without dysplasia and in the normal oral epithelium, three fields were randomly selected to determine average ratio of a positive cell. In the Wnt3 staining, the cell membranous or cytoplasmic staining in the basal and spinous layer was regarded as positive. For cyclin D1 and c-myc, nuclear staining was regarded as positive. The cyclin D1 and c-myc staining was evaluated by counting at 200-fold magnification and calculating the percentage of positive cells. Statistical significance was estimated using Fisher's exact test, Mann-Whitney GSK-3 – glycogen synthase kinase-3; LEF1 – lymphoid enhancer binding factor 1; LRP – low-density lipoprotein receptor-related protein; OSCC – oral squamous cell carcinoma; PBS – phosphate-buffered saline; TCF – T cell factor.The author(s) declare that they have no competing interests.KI and SI carried out immunohistochemical analysis and evaluation of staining, KI also drafted the manuscript, NW participated in evaluation of immunohistochemical staining and helped to draft the manuscript, HD and TH participated to collect specimens and performed in the statistical analysis, MH and TN participated in the design and coordination of the study, TN also conceived of the study and helped to draft the manuscript. All authors read and approved the final manuscript.


Adherens junctions and desmosomes are intercellular adhesive junctions and essential for the morphogenesis, differentiation, and maintenance of tissues that are subjected to high mechanical stress, including heart and skin. The different junction complexes are organized at the termini of the cardiomyocyte called the intercalated disc. Disruption of adhesive integrity via mutations in genes encoding desmosomal proteins causes an inherited heart disease, arrhythmogenic right ventricular cardiomyopathy (ARVC). Besides plakoglobin, which is shared by adherens junctions and desmosomes, other desmosomal components, desmoglein-2, desmocollin-2, plakophilin-2, and desmoplakin are also present in ultrastructurally defined fascia adherens junctions of heart muscle, but not other tissues. This mixed-type of junctional structure is termed hybrid adhering junction or area composita. Desmosomal plakophilin-2 directly interacts with adherens junction protein alphaT-catenin, providing a new molecular link between the cadherin-catenin complex and desmosome. The area composita only exists in the cardiac intercalated disc of mammalian species suggesting that it evolved to strengthen mechanical coupling in the heart of higher vertebrates. The cross-talk among different junctions and their implication in the pathogenesis of ARVC are discussed in this review. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart muscle disease estimated to affect approximately 1 in 5,000 individuals –4. ApproDesmosomes and adherens junctions are intercellular adhesive junctions that anchor intermediate filaments and actin cytoskeleton, respectively, at the plasma membrane of adjoining cells, thereby provide mechanical attachment between the cells, and support the structural and functional integrity of the tissues. Desmosomes consist of three families of proteins, desmosomal cadherins, armadillo proteins, and plakins  7]. Des. Des7]. β-catenin or PG. β-catenin or PG links cadherins to α-catenin, and α-catenin interacts with the actin cytoskeleton. Gap junction is another intercellular junction in the cell responsible for cell-cell communication and electrical coupling by mediating small molecules and ion transfer between the cells. Each gap junction is composed of two hexameric structures called connexons or hemichannels that dock across the extracellular space and form a permeable pore. Each connexon consists of six transmembrane proteins called connexin (Cx). Cx43 is the most abundant connexin isotype in the heart. The different junctional complexes must be properly organized in the intercalated disc (ICD) of the myocardium to preserve normal mechanical and electrical function of the heart.The classical cadherins are calcium-dependent cell adhesion receptors, located in the adherens junction. The extracellular domain of cadherins interacts homophilically mediating strong cell-cell adhesion and plays a key role in the maintenance of tissue structure. In the classic model for adherens junction, the cytoplasmic tail of cadherin interacts in a mutually exclusive manner with either γ-catenin, is the only linker protein present in both desmosomes and adherens junctions in skin and heart [JUP) in affected individuals [JUP mutation (S39_K40insS) to cause ARVC without cutaneous abnormalities [Plakoglobin, also known as nd heart . Plakoglividuals . A studymalities . Importamalities . Most remalities . The mixing of junctional components in the heart was first described in PG null mice. The homozygous PG null animals die between embryonic days 12–16 due to ventricular rupture and hemorrhaging into the pericardial cavity. Desmosomes are not detected in the mutant hearts. Instead extended adherens junctions develop, which contain desmosomal proteins such as desmoplakin, forming “mixed type” adhering junction , 15. In The typical morphological appearance of cardiac intercalated disc at the ultra structural level in the mice is submembranous plaques with electron dense material adjacent to intercellular space between the myocytes . A relatα-catenin, and β-catenin, also have been shown to colocalize with desmosomal proteins in the majority of the area composita junctions [By comprehensive immunoelectron microscopy with immunogold DP antibody labeling, Franke et al. observed that in normal heart muscle, DP is located in all plaques of both the desmosome-like and fascia adhaerens-type junctions. Very intensely labeled junctions with DP are seen in the more desmosome-like junctions , whereasunctions . These sunctions . Consistunctions . By contunctions , 21. αT-catenin is a recently identified member of α-catenin family with restricted expression in testis, brain, and cardiac muscle [αT-catenin, a unique molecule of adherens junction, functions as cytoskeletal linker protein that specifically brings the desmosomes and adherens junctions together in the intercalated disc of the heart [αT-catenin was shown to interact specifically with desmosomal PKP-2 implicating a novel molecular linkage between the adherens junction and desmosome. By double αT-catenin/PKP-2 immunolabeling electron microscopy, αT-catenin is observed to colocalize with other molecules of cadherin/catenin complex, β-catenin, and N-cadherin at the fascia adhaerens-like junctions, but also with desmosomal proteins such as PKP-2, DSG-2, and DP. By contrast, no localization of either αE-catenin or β-catenin could be seen at desmosome-like junctions of the ICD. Based on the biochemical and morphological studies, αT-catenin is thought to recruit desmosomal proteins to hybrid adhering junctions, forming a mixed-type, reinforced junction at the ICD that is attached to both the intermediate filaments and actin cytoskeletons  [CTNN3 mutations to date, αT-catenin is considered a candidate gene and may be the potential cause of DCM or ARVC [c muscle . Extensihe heart . Using ykeletons . Accordirhythmia . αT-catehy (DCM) . Althoug or ARVC . It is estimated that as many as 70% of the desmosomal mutations linked to familial ARVC are in the gene coding for PKP-2 . AbnormaThe presence of stable mechanical coupling mediated by N-cadherin/catenin adhesion complex is of paramount importance for maintaining the structural integrity of the heart. We demonstrated that cardiac-specific deletion of N-cadherin in mice (N-cad CKO) causes dissolution of the adherens junction, desmosomes, and area composita resulting in absence of ICD structure in the N-cadherin mutant heart . Gap junαT-catenin/PKP-2 and PKP-2/Cx43. The different junctional complexes must be properly localized in the ICD to mediate normal mechanical and electrical coupling between cardiomyocytes. The mixed-type, reinforced junction at the ICD may have evolved to maintain the exceptionally large gap junction plaques found in the mammalian heart, which is under high mechanical stress. Given the large number of PKP-2 mutations identified in ARVC patients, it will be imperative to understand the function of this plakophilin in maintaining electrical synchrony in the heart. Recent studies from human genetics and animal models suggest cross-talk between intercellular junctions constituting the ICD, including adherens junction, desmosome, area composita, and gap junctions. Loss-of-function studies in mice have provided important insight into the hierarchical relationship between the different junction complexes; however subtle mutations in ICD proteins will be necessary to understand the molecular mechanisms underlying ARVC. In the future, it will be interesting to know how the area composita is affected in ARVC patients and its implication in the pathogenesis of the disease.In conclusion, the idea that anchoring junctions of the ICD are segregated into distinct domains performing independent functions needs to be reevaluated based on recent data demonstrating novel protein interactions between components from different junction complexes; for example,