Proteolysis of huntingtin (Htt) plays a key role in the pathogenesis of Huntington's disease (HD). However, the environmental cues and signaling pathways that regulate Htt proteolysis are poorly understood. One stimulus may be the DNA damage that accumulates in neurons over time, and the subsequent activation of signaling pathways such as those regulated by IκB kinase (IKK), which can influence neurodegeneration in HD.We asked whether DNA damage induces the proteolysis of Htt and if activation of IKK plays a role. We report that treatment of neurons with the DNA damaging agent etoposide or γ-irradiation promotes cleavage of wild type (WT) and mutant Htt, generating N-terminal fragments of 80–90 kDa. This event requires IKKβ and is suppressed by IKKα. Elevated levels of IKKα, or inhibition of IKKβ expression by a specific small hairpin RNA (shRNA) or its activity by sodium salicylate, prevents Htt proteolysis and increases neuronal resistance to DNA damage. Moreover, IKKβ phosphorylates the anti-apoptotic protein Bcl-xL, a modification known to reduce Bcl-xL levels, and activates caspases that can cleave Htt. When IKKβ expression is blocked, etoposide treatment does not decrease Bcl-xL and activation of caspases is diminished. Similar to silencing of IKKβ, increasing the level of Bcl-xL in neurons prevents etoposide-induced caspase activation and Htt proteolysis.These results indicate that DNA damage triggers cleavage of Htt and identify IKKβ as a prominent regulator. Moreover, IKKβ-dependent reduction of Bcl-xL is important in this process. Thus, inhibition of IKKβ may promote neuronal survival in HD as well as other DNA damage-induced neurodegenerative disorders. Huntington's disease is a neurodegenerative disorder caused by expansion of a CAG repeat, which is translated into a polyglutamine (polyQ) stretch in the N-terminus of Htt protein Drosophila polyQ model DNA double-stranded breaks progressively accumulate in the aging brain, and elevated DNA damage is found in HD patients and HD animal models DNA damage is a potent inducer of IKK We previously showed that HD mouse models have elevated IKKβ/NF-κB in the CNS, and blocking IKKβ activity prevents degeneration of medium-sized spiny neurons caused by a toxic fragment of mutant Htt In this study we used a human embryonic neuronal stem cell line (MESC2.10) isolated from the midbrain of an eight-week-old embryo in vitro kinase assays using recombinant IκBα as the substrate for IKKβ complexes immunoprecipitated from the extracts of etoposide-treated neurons. We find that IKKβ is activated 2 hrs after etoposide treatment and remains active for up to 4 hrs , indicating that Bcl-xL reduction is likely due to enhanced protein turnover. Although phosphorylation-induced degradation of Bcl-xL in the presence of genotoxic agents is a necessary step for induction of apoptosis, it is unclear which kinase(s) mediates this event s Bcl-xL .Q111/Q111) Generation of N-terminal fragments of mutant Htt is thought to initiate neurotoxicity, culminating in HD The novel aspects of present work are the triggering of Htt proteolysis by DNA damage and the identification of IKKβ as a prominent regulator of enzymes known to cleave Htt. We propose that in post-mitotic neurons, IKKβ activation by DNA damage promotes Htt cleavage by influencing Bcl-xL and a caspase pathway. Conversely, our data support a protective role for IKKα, whose expression in neurons prevents DNA damage-induced Htt proteolysis .Although acute IKKβ activation is known to be predominantly pro-survival, under certain circumstances it promotes apoptosis and neurodegeneration in vivo is worthy of investigation in HD animal models.In contrast to IKKβ, IKKα promotes neuronal survival and prevents Htt proteolysis. This could simply result from suppression of IKKβ activity by IKKα  [23]. HoThe mechanism of Htt proteolysis is complex and probably involves integration of multiple signaling pathways. Our results identify Bcl-xL as a potential mediator of DNA damage-induced Htt proteolysis . Bcl-xL Environmental factors appear to play a role in the development of HD, since the age of onset is variable among patients with similar polyQ expansions Anti-Htt (mAb 2166), and PSD-95 antibodies were purchased from Millipore . Antibodies recognizing IKKγ, IKKβ, caspase-3 and caspase 6 were purchased from Cell Signaling Technology . Anti-IKKα was purchased from BD Biosciences . Anti-Bcl-xL and anti-p53 antibodies were from Santa Cruz Biotechnologies . Anti-Tuj-1 antibody was purchased from Covance . Cell fractionation and ECL detection kits including HRP-conjugated secondary antibodies were from PIERCE Biotechnology . Anti-γ-H2AX antibody, caspase-3 and caspase-6 activity kits, and recombinant Bcl-xL protein were obtained from R&D systems . Anti-laminB1 antibody, DMEM/F12, N-2 and B-27 media supplements were purchased from Invitrogen . Fibroblast-growth factor-2 (FGF-2) was obtained from Promega . Sodium salicylate, etoposide, and caspase-3 and caspase-6 inhibitors , and anti-tubulin antibody were purchased from Sigma/Aldrich . Recombinant active IKKα and IKKβ were purchased from Upstate Biotechnologies . Consensus NF-κB oligonucleotides for p65 and p52 coated onto 96 wells and the corresponding antibodies for binding detection (TransFactor NF-κB p65 and p52) were purchased from Clontech The methods for generation and differentiation of MESC2.10 human neuroblasts have been reported previously Q111/Q111, obtained from homozygous knock-in mice were propagated as described Mouse striatal neuronal precursors, HdhQ111/Q111) were treated with etoposide and analyzed as described for MESC2.10 cells.MESC2.10 cells, differentiated for 9 days, were treated with 10 µM etoposide for the periods indicated in each figure. Neurons were harvested and separated into cytoplasmic and nuclear fractions using the NE-PER kit (PIERCE) according to instructions. For most experiments we used ∼120 µg of lysate for SDS-PAGE and Western blotting with the indicated antibodies. Reactive bands in Western blots were detected by enhanced chemiluminescence (ECL) using a gel documentation system. Sodium salicylate (5 mg/ml), caspase-3 inhibitor (Ac-DEVD-CHO) and casapse-6 inhibitor (Ac-VEID-CHO) were added at 20 µM 1 hr prior to etoposide treatment. To examine the effects of etoposide on mutant Htt differentiated mouse striatal cells  (1∶500) (R&D systems). Anti-Tuj-1 was used to label the cytoplasm (1∶1000). Goat anti-rabbit conjugated to FITC (green) and goat anti-mouse conjugated to rhodamine (red) was used as secondary antibodies. Pictures were captured with a confocal microscope.IKKα was cloned into the lentiviral FUGW vector under the control of a ubiquitin promoter 32P-γ-ATP for 30 min at 30°C. GST protein was used as a negative control. All kinase products were examined by SDS-PAGE and autoradiography.IKKβ activity was assayed as described previously Nuclear extracts for control or etoposide-treated MESC2.10 neurons were obtained using the cell fractionation kit from pierce according to the instructions provided. Fifty µg of each nuclear extract was incubated for an hour at RT on 96 well plates  coated with consensus NF-κB oligonucleotides (p65 or p52). Mutated NF-κB oligonucleotides were used to confirm binding specificity. For competition assays, nuclear extracts were pre-incubated with NF-κB oligonucleotides for 1 hr on ice and then added to the coated wells. After washing, each well was incubated with anti-p65 or anti-p52 antibodies for 30 min at 37°C. Wells were washed as recommended by the manufacturer (Clontech), followed by incubation with a secondary antibody conjugated to HRP. TMB  was added for 10 min. Binding was measured in a microplate reader at 655 nm.To measure the activity of caspase-3 or -6, the colorimetric assay kits from R&D Systems were used according to manufacturer's instructions. Briefly, MESC2.10 neurons were pre-incubated with caspase-3 inhibitor (Ac-DEVD-CHO), casapse-6 inhibitor (Ac-VEID-CHO) or sodium salicylate (5 mg/ml) 1 hr prior to etoposide treatment, which was for an additional 6 hrs. Cells were lysed as instructed. Fifty µg of each lysate was incubated with either caspase-3 substrate  or caspase-6 substrate  in a 96 well plate at 37°C for 1 hr. Enzyme activities for caspase-3 or -6 were measured with a microplate reader at 405 nm. Results are shown as relative enzyme activity and represent average of three experiments.Figure S1γ-irradiation induced DNA damage promotes Htt cleavage in MESC2.10 neurons. γ-irradiation was carried out using a MARK-I γ-irradiator with a 137Cs source at a specific dose rate of 1.22 Gy/min. Cells were irradiated with 5 Gy and further incubated for the indicated time. Sodium salicylate  was added at a concentration of 5 mg/ml 1 hr prior to irradiation and incubated for 6 hr. Etoposide treatment (10 µM) was used as a positive control and was carried out for 6 hr (lane 6). Lanes 7 and 8 represent MESC2.10 neurons transduced with an IKKβ shRNA lentivirus  and irra(4.20 MB TIF)Click here for additional data file.


Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease, characterized by distorted lung architecture and loss of respiratory function. Alveolar epithelial cell injury and hyperplasia, enhanced extracellular matrix deposition, and (myo)fibroblast activation are features of IPF. Wnt/β-catenin signaling has been shown to determine epithelial cell fate during development. As aberrant reactivation of developmental signaling pathways has been suggested to contribute to IPF pathogenesis, we hypothesized that Wnt/β-catenin signaling is activated in epithelial cells in IPF. Thus, we quantified and localized the expression and activity of the Wnt/β-catenin pathway in IPF.in vitro studies further revealed that Wnt ligands induced lung epithelial cell proliferation and (myo)fibroblast activation and collagen synthesis.The expression of Wnt1, 3a, 7b, and 10b, the Wnt receptors Fzd1-4, Lrp5-6, as well as the intracellular signal transducers Gsk-3β, β-catenin, Tcf1, 3, 4, and Lef1 was analyzed in IPF and transplant donor lungs by quantitative real-time (q)RT-PCR. Wnt1, 7b and 10b, Fzd2 and 3, β-catenin, and Lef1 expression was significantly increased in IPF. Immunohistochemical analysis localized Wnt1, Wnt3a, β-catenin, and Gsk-3β expression largely to alveolar and bronchial epithelium. This was confirmed by qRT-PCR of primary alveolar epithelial type II (ATII) cells, demonstrating a significant increase of Wnt signaling in ATII cells derived from IPF patients. In addition, Western blot analysis of phospho-Gsk-3β, phospho-Lrp6, and β-catenin, and qRT-PCR of the Wnt target genes cyclin D1, Mmp 7, or Fibronectin 1 demonstrated increased functional Wnt/β-catenin signaling in IPF compared with controls. Functional Our study demonstrates that the Wnt/β-catenin pathway is expressed and operative in adult lung epithelium. Increased Wnt/β-catenin signaling may be involved in epithelial cell injury and hyperplasia, as well as impaired epithelial-mesenchymal cross-talk in IPF. Thus, modification of Wnt signaling may represent a therapeutic option in IPF. Pulmonary fibrosis can result from a variety of causes, including lung injury, environmental particle and toxin inhalation, chemotherapy, systemic autoimmune diseases, or as an idiopathic entity in form of idiopathic interstitial pneumonias (IIP) Fibroblast foci represent the hallmark lesions of IPF, as they constitute aggregates of activated myofibroblasts, which promote excessive ECM deposition The Wnt family constitutes a large family of highly conserved secreted growth factors essential to organ development, a process often recapitulated in organ failure. The best characterized Wnt signaling pathway is the β-catenin-dependent, or canonical, Wnt signaling pathway Importantly, increased nuclear β-catenin staining was recently reported in IPF tissue sections Initially, we sought to quantify the mRNA expression of canonical Wnt/β-catenin signaling components in lung tissue samples of transplant donors and IPF patients using quantitative real-time (q)RT-PCR. As depicted in Next, we analyzed the expression of common Wnt receptors and co-receptors. As shown in The main canonical Wnt signal transducers Gsk-3β and β-catenin were both expressed in normal and fibrotic lung tissue, with a significantly increased expression of β-catenin in IPF (log-fold change 0.98±0.28) . With thWe went on to localize cell types capable of Wnt ligand secretion, as assessed by immunohistochemistry of Wnt1 and 3a ligands, and cell types capable of Wnt signaling, as assessed by immunohistochemistry of β-catenin and Gsk-3β, in donor and IPF lung tissue , 3, 4, 5We then analyzed protein expression pattern of β-catenin and Gsk-3β . ImportaTo further corroborate these results, we quantified cell-specific gene expression of Wnt/β-catenin signaling components in primary human ATII cells and fibroblasts derived from IPF patients or transplant donors, using qRT-PCR . As depiTo accurately assess, whether the Wnt/β-catenin signaling pathway was activated in IPF, we performed Western blot analysis of phospho-Gsk-3β, phospho-Lrp6, and β-catenin. As presented in 3±28×103 versus 119×103±16×103 for Wnt3a and control, respectively; We then sought to investigate biological effects elicited by Wnt ligands on key cell types involved in IPF pathogenesis. To this end, we assessed proliferation, as well as (myo)fibroblast activation and collagen deposition in A549 lung epithelial cells und NIH-3T3 fibroblasts, respectively. Using a Tcf/Lef-driven reporter gene assay, we first demonstrated that Wnt3a elicited a potent canonical Wnt/β-catenin response, while Wnt7a did not  and fibroblast-specific protein (Fsp) 1, as assessed by qRT-PCR of Wnt3a- or vehicle-treated fibroblasts . This coIPF is the most common form of the idiopathic interstitial pneumonias. IPF exhibits a poor prognosis and unresponsiveness to currently available therapies, reflecting our limited understanding of the basic mechanisms and mediators implicated in the pathogenesis of this progressive and fatal disease In this respect, the Wnt signaling system is of particular interest, as it constitutes a developmentally active pathway, which is reactivated in chronic diseases characterized by pathologic tissue remodeling To this end, we performed a comprehensive analysis of canonical Wnt signaling components at the mRNA and protein level, assessing expression, localization, as well as activity of Wnt ligands, receptors, and intracellular signaling molecules in IPF. We demonstrate that all essential components were expressed in the human lung, and particularly localized to the alveolar and bronchial epithelium in normal, as well as IPF lungs, as demonstrated by qRT-PCR and immunohistochemistry. Lung homogenate, as well as cell-specific analysis of Wnt ligands and receptors demonstrated increased expression of Wnt ligands compare  and 6, wSimilar to the Wnt ligands, Gsk-3β and β-catenin were highly expressed in hyperplastic ATII cells and bronchiolar epithelial cells at sites of bronchiolization in IPF. This suggests that Wnt signaling, which is known to drive epithelial cell hyperplasia in non-pulmonary epithelia Our current understanding about the functional relevance of Wnt signaling in lung epithelium is largely derived from transgenic animal models. If deleted by homologous recombination in mice, Wnt5a deficiency leads to overexpansion of distal airways and inhibition of lung maturation, accompanied by enlarged intersaccular interstitial compartments in vitro studies, such as Wnt1, are commonly either overexpressed by viral transduction, or supplied in the form of conditioned media harvested from Wnt1 overexpressing cell lines In this study, we used recombinant Wnt3a to assess the functional effects of canonical Wnt signaling in epithelial cells and fibroblasts. While we observed decreased expression of Wnt3a in IPF homogenates and ATII cells  and 6, wOne of the key questions arising from this study is whether different Wnt isoforms, such as Wnt1 and Wnt3a, are able to induce different effects, or whether they are merely expressed in a distinct spatio-temporal fashion in the lung to elicit different phenotypes, if deleted in mice. In general, a specific biological effect induced by Wnt ligand exposure depends on the expression of distinct Wnt receptors and signaling molecules, distinct ECM molecules such as glypicans, but also on the presence of Wnt inhibitors, such as Dickkopfs, secreted frizzled receptor proteins, or Wnt inhibitory factor We also demonstrated increased levels of β-catenin in IPF, which were predominantly localized to bronchial epithelial and hyperplastic ATII cells. In contrast to a previous investigation presenting increased nuclear β-catenin staining in bronchial epithelial and ATII cells, as well as interstitial fibroblasts in vitro and in vivo studies will undoubtedly shed more light on the mechanistic principles underlying Wnt activation in IPF, and whether therapeutic modulation thereof will present as a suitable therapeutic tool in this devastating disease.In sum, we report increased functional Wnt signaling in IPF, documented by increased phosphorylation of Lrp6 and Gsk-3β, which has recently been demonstrated to present as a most sensitive indicator of Wnt activity in tissue sections The following antibodies were used in this study: Total β-catenin (#9562), phospho-S9- and total Gsk-3β , phospho- and total Lrp6 , Wnt1 , Wnt3a , CyclinD1 , α-smooth muscle actin , collagen type 1  and Lamin A/C . Dulbecco's modified Eagle's medium (DMEM) and fetal calf serum (FCS) were obtained from Invitrogen. Recombinant Wnt3a and Wnt7a was purchased from R&D Systems.CO/VA = 23%±3%; O2 = 2–4 l/min; PaO2 = 49–71 mmHg, PaCO2 = 33–65 mmHg) and 9 control subjects . Individual patient characteristics are shown in Lung tissue biopsies were obtained from 15 IPF patients with histological usual interstitial pneumonia (UIP) pattern  cells were isolated, as previously described Hprt1 and Pbgd, ubiquitously and equally expressed genes free of pseudogenes, were used as a reference gene in all human and mouse qRT-PCR reactions, respectively. PCR was performed using the primers listed in reference – Cttarget). Relative changes in transcript levels compared to controls are ΔΔCt values (ΔΔCt = ΔCttreated – ΔCtcontrol). All ΔΔCt values correspond approximately to the binary logarithm of the fold change as mentioned in the text. When relative transcript abundance is of information, expression levels are given in ΔCt levels.Total RNA was extracted using Qiagen extraction kits according to the manufacturer's protocol, and cDNAs were generated by reverse transcription using SuperScript™ II (Invitrogen) 2O2 for 20 min. Immune complexes were visualized using suitable peroxidase-coupled secondary antibodies, according to the manufacturer's protocol  Human lungs were placed in 4% (w/v) paraformaldehyde after explantation, and processed for paraffin embedding. Sections (3 µm) were cut, mounted on slides, subjected to antigen retrieval, and quenching of endogenous peroxidase activity using 3% (v/v) HNIH-3T3 cells were plated on chamber slides, fixed with acetone/methanol (1∶1), and blocked for unspecific binding sites with 3% (m/vol) BSA. Fixed cells were incubated with the indicated primary antibodies for 60 min in PBS containing 0.1% (m/vol) BSA. Indirect immunofluorescence was performed by incubation with Alexa 555-conjugated secondary antibodies . Nuclei were visualized by 4,6-diamidino-2-phenylindole staining .Human lung tissue specimens were homogenized in extraction buffer  and whole proteins were extracted by centrifugation (12.000× g) for 10 min at 4°C, as described previously Lung epithelial A549 cells were transiently transfected with the reporter construct TOPflash or FOPflash  using Lipofectamine™ 2000 (Invitrogen). The TOPflash construct contains eight Tcf/Lef binding sites upstream of a minimal TA viral promoter and the firefly luciferase cDNA. The FOPflash construct is identical with the exception that it contains mutated copies of TCf/Lef binding sites and is used as a control for measuring nonspecific activation of the reporter construct. Treatment with Wnt3a (100 ng/ml) or Wnt7a (100 ng/ml) for 24 h was performed 4 h after transfection. Luciferase activities were determined using the Dual Luciferase Assay System (Promega) on a Fusion™ luminometer (Packard BioScience).NIH-3T3 fibroblasts were plated at a density of 30.000 cells/well in 6-well plates, synchronized for 24 h in serum-free medium, and treated for 24 h as indicated. Total collagen content was determined using the Sircol Collagen Assay kit . Equal amounts of protein lysates were added to 1 ml of Sircol dye reagent, followed by 30 min of mixing. After centrifugation at 10.000× g for 10 min, the supernatant was carefully aspirated and 1 ml of Alkali reagent was added. Samples and collagen standards were then read at 540 nm on a spectrophotometer (Bio-Rad). Collagen concentrations were calculated using a standard curve generated by using acid-soluble type 1 collagen.t-test, or a one-way analysis of variance (ANOVA) with Tukey HSD post hoc test for studies with more than 2 groups. All p values obtained from multiple tests were adjusted using the procedure from Hochberg and Benjamini All ΔCt values obtained from real-time RT-PCR were analyzed for normal distribution using the Shapiro-Wilk test, using assignment of a normal distribution with p>0.05. Normality of data was confirmed using quantile-quantile plots. The means of indicated groups were compared using two-tailed Student's


We have investigated how Wnt and vitamin D receptor signals regulate epidermal differentiation. Many epidermal genes induced by β-catenin, including the stem cell marker keratin 15, contain vitamin D response elements (VDREs) and several are induced independently of TCF/Lef. The VDR is required for β-catenin induced hair follicle formation in adult epidermis, and the vitamin D analog EB1089 synergises with β-catenin to stimulate hair differentiation. Human trichofolliculomas (hair follicle tumours) are characterized by high nuclear β-catenin and VDR, whereas infiltrative basal cell carcinomas (BCCs) have high β-catenin and low VDR levels. In mice, EB1089 prevents β-catenin induced trichofolliculomas, while in the absence of VDR β-catenin induces tumours resembling BCCs. We conclude that VDR is a TCF/Lef-independent transcriptional effector of the Wnt pathway and that vitamin D analogues have therapeutic potential in tumors with inappropriate activation of Wnt signalling. Adult mammalian epidermis is maintained by stem cells that self-renew and produce progeny that differentiate along the lineages of the hair follicle (HF), sebaceous gland (SG) and interfollicular epidermis (IFE) β-catenin interactions with Lef1 and Tcf3 in the epidermis have been extensively characterised There is evidence for both ligand dependent and independent effects of VDR. In cultured cell lines β-catenin can bind to unligated VDR, but complex formation is enhanced by vitamin D, and β-catenin stimulates vitamin D dependent transcription It has recently been reported that VDR ablation leads to a gradual decrease in epidermal stem cells, consistent with postnatal hair loss Given the uncertainty regarding the role of VDR ligands in hair follicle maintenance, we began by examining the conditions under which VDR-β-catenin complexes formed in primary mouse epidermal keratinocytes. In immunoprecipitation/Western blot assays we could not detect complex formation in the absence of Wnt3A and EB1089, or when cells were treated with a single ligand. Complex formation was only detectable when cells were stimulated with both Wnt3A and EB1089 . EB1089 We next examined how the canonical Wnt and vitamin D pathways interacted to regulate transcription. We transfected primary mouse keratinocytes with luciferase reporters of transcriptional activity: a TOPFlash reporter containing 3 TCF/Lef binding sites and a VDRE reporter consisting of 4 vitamin D response elements (4×VDRE) . Wnt3A iWnt3A strongly enhanced induction of the vitamin D response construct by EB1089 in wild type and ΔNLef1 cells . TransieWe next examined whether β-catenin acted as a transcriptional co-activator of VDR on a natural promoter. Keratin 15 is expressed by stem cells in the HF bulge and in β-catenin induced ectopic HF in vivo, we performed a systematic analysis of the promoter regions of genes previously reported to be upregulated by 4-hydroxy-Tamoxifen (4OHT) treatment of the skin of K14ΔNβ-cateninER transgenic mice To investigate the significance of β-catenin/VDR interactions Many of the genes upregulated by β-catenin  encode pIn order to characterise the transcription factor complexes binding to the promoters of these genes, we performed ChIP on three of them . We desiThe PADI3 promoter differed from the S100A and K15 promoters because it had no TCF/Lef binding sites . NeverthAs further confirmation that β-catenin is recruited through the VDR to region 3 of the PADI3 and K15 promoters, we performed ChIP in VDR null primary mouse keratinocytes . WhereasWe conclude that β-catenin bound and enhanced ligand activated VDR in Wnt target genes, both in presence of Lef1 (K15 and S1003A) and independently of Lef1 (PADI3).in vivo in adult epidermis, we tested the effect of EB1089 on 4OHT treated K14ΔNβ-cateninER transgenic mice from two founder lines, D2 and D4. Activation of β-catenin stimulates proliferation and anagen entry in both high (D4) and low (D2) transgenic copy number mouse lines To examine whether β-catenin and the VDR also cooperated to promote hair follicle differentiation When D4 transgenic mice were treated with 4OHT for 14 days, existing HFs enlarged and entered anagen and K17, which is normally confined to the ORS , was expMany of the genes identified as transcriptional targets of β-catenin/VDR complexes encode hair follicle proteins , leadingThe effects of EB1089 were not accompanied by significant changes in proliferation in either the D2 or the D4 line, whether visualised by Ki67 labelling of epidermal whole mounts  or by flIt has previously been reported that in VDR−/− epidermis there is a gradual decrease in the size of the stem cell compartment and that this correlates with a failure of β-catenin to induce proliferation required for anagen entry VDR heterozygous mice were indistinguishable from wild type . AlthougK14ΔNβ-cateninER mice that were heterozygous for VDR were indistinguishable from D2 mice on a wild type background: 4OHT induced existing follicles to enter anagen, resulting in lengthening of the hairs on the tail , and ectThe inhibition of β-catenin induced hair follicle differentiation did not correlate with a failure of VDR −/− epidermis to proliferate in response to β-catenin. The proportion of proliferative cells, detected by Ki67 labelling or flow cytometry, was higher in VDR null than wild type epidermis, and the increase in proliferation on β-catenin activation was very similar in both VDR+/− and VDR−/− backgrounds . InductiIf VDR/β-catenin interactions stimulate HF differentiation, then EB1089 may inhibit β-catenin induced tumour formation. Prolonged activation of β-catenin signalling in the D4 line of K14ΔNβ-cateninER transgenic mice results in the conversion of almost all of the follicles into benign tumours known as trichofolliculomas If the primary effect of VDR loss is to deplete the stem cell compartment To investigate whether or not nuclear β-catenin and VDR expression correlated with tumour type in human skin tumours, we examined a panel of 59 human tumours, which were categorised as basal cell carcinomas or having elements of HF differentiation (trichofolliculomas (TF), trichoepithelioma (TE), or sebaceous TF (STF) . Those tOur results are summarised schematically in It has recently been reported that the mechanism by which VDR loss leads to postnatal alopecia is via progressive stem cell depletion β-catenin can no longer be considered as chiefly an activator of TCF/Lef target genes In human skin tumours with detectable nuclear β-catenin, the level of VDR correlates with differences in tumour phenotype, a similar situation to human colon cancer The antibodies used have been described previously Primary keratinocytes derived from wild type, VDR null and K14ΔNLef1 mice TCF/Lef and VDR variant consensus motifs were defined by comparing the natural binding sites in the promoter region of several target genes . The sitIn evaluating human tumour sections we calculated the probability of infiltrative tumours having low levels of VDR and detectable levels of nuclear β-catenin and performed a contingency analysis using the ChiSquare Test .The D2 and D4 lines of K14ΔNβ-cateninER transgenic mice have been described previously The K14ΔNβ-cateninER transgene was activated by topical application of 4-hydroxytamoxifen  dissolved in acetone. Tail skin was treated by applying 4OHT with a paint brush (0.5 mg per mouse every second day). In some experiments, mice received topical applications of EB1089 (1.5 mg in acetone per mouse) 30 min prior 4OHT treatment. All animal experiments had the approval of the CR-UK London Research Institute ethics committee and were carried out under a British Home Office license.Three independent experiments comparing D4 line of K14ΔNβ-cateninER transgenics and transgenic negative littermates (wild type) were performed. The combined total of mice per treatment group and genotype was 16.Four independent experiments examining the D2 line of K14ΔNβ-cateninER of transgenic mice on a VDR−/+ or VDR−/− backgrounds, were performed. A total of 18 mice per treatment group/genotype was examined.−7 M EB1089 and/or 100 ng/ml Wnt3A. In some experiments cells were cotransfected with pSG5-VDR or pSG5 (Mock) constructs. Luciferase activity was measured using the PerkinElmer EnVision™ system.Primary mouse keratinocytes were grown in KSFM medium (Invitrogen-Gibco) and deprived of serum overnight prior to treatment. Cell lysis, immunoprecipitation and immunoblotting were performed as described 5′tttttttttttttt 3′), 0.5 mM dNTP mix, 0.05 mM DTT, 4 µl of 5× First Strand Buffer (Invitrogene) and 1 µl of Retrotranscriptase (Invitrogene) in a final 20 µl reaction. All samples where analyzed by real-time PCR using SYBR Green PCR master mix (Applied Biosystems) and specific pairs of primers for each gene . Retrotranscription of mRNA to cDNA was performed using 0.5 µg of total RNA from each sample, 2.5 µM oligo dT primer (ach gene .−7 M EB1089 and/or 100 ng/ml Wnt3A for 4 h. 106 keratinocytes were lysed and immunoprecipitated with 2.5 µg of VDR (Sigma), β-catenin (BD Transduction), Lef1 (Upstate Biotech) or unrelated HA (Roche) antibodies. Sonication, precipitation and washes were performed using an Upstate ChIP assay kit. All the samples where analysed by real-time PCR using SYBR Green PCR master mix (Applied Biosystems) and specific pairs of primers for two regions of each promoter that contained VDRE and/or TCF/Lef binding sites, one region that does not contain any of these sites, and a completely unrelated region of the genome as a negative control  was logarithmic. The point at which the fluorescence crossed the threshold is called the Ct. We corrected the Ct values of each sample by the Ct value obtained for the corresponding input using the same primers or using unrelated primers. The formula used is shown as follows:a : Ct value for PCR of immunoprecipitated samples.i : Ct value for PCR of imput samples (prior immunoprecipitation).c : Ct value for PCR using unrelated oligos for each sample.DctIP : Differential Ct for the immunoprecipitates. DctIP = a-iDctC : Differential Ct control. DctC = c-iDDct : Differential between DctIP and DctC. DDct = DctIP-DctC.DDctFold = 2Conventional fixed, paraffin-embedded sections and tail epidermal whole mounts were prepared and immunolabeled as described previously To analyze cell cycle keratinocytes isolated from epidermis were fixed with 2% paraformaldehyde (PFA) at room temperature for 10 min and then permeabilized with 0.1% Triton-X100 for 10 min. Cells were washed with PBS and re-suspended in PBS with 2% FBS containing DAPI . Pulse processing was used in order to exclude any unstained, apoptotic or clumped cells and analysis was performed using an LSRII (Becton-Dickinson). Analysis of flow cytometry data was performed using a FlowJo 6.3.3 .Figure S1Expression of beta-catenin target genes in wild type anagen follicles. (A) H&E staining. Positions of dermal papilla (DP), hair matrix (ma), outer root sheath (ORS), inner root sheath (IRS) and hair shaft (HS) are indicated. (B-L) Immunostaining with antibodies indicated (green), anti-keratin 14 (red) and Hoescht (blue) counterstain. Scale bar: 100 micrometers.(9.52 MB TIF)Click here for additional data file.Figure S2Beta-catenin is recruited to hair follicle gene promoters by binding ligand activated VDR.  Wild type  and VDR null  primary mouse keratinocytes were lysed and immunoprecipitated with VDR, beta-catenin or unrelated antibody . Immunoprecipitated genomic DNA fragments or input controls were amplified by real-time PCR using specific primers for region 3 of the mouse promoters indicated or unrelated genomic primers (UR). Data are means±S.D. of triplicate reactions. Scaled diagrams summarize location of VDREs and TCF/Lef binding sites and the proteins bound to each region in cells treated with Wnt3A and EB1089.(0.91 MB TIF)Click here for additional data file.Figure S3EB1089 promotes ectopic hair follicle differentiation without affecting proliferation in K14DeltaNbeta-cateninER transgenic mice. Epidermal sections (A–F) or whole mounts (G–H) of D4 tail skin treated with 4OHT and/or EB1089. (A–D) Double immunolabelling with keratin 14 (red) and the antibodies shown (green), with Hoechst (blue) counterstain. Asterisks indicate ectopic HFs encircling dermal papillae. Dashed lines demarcate dermal-epidermal boundary.  Alkaline phosphatase activity (blue) with fast red counterstain. Asterisk indicates dermal papilla. (G–I) Ki67 staining (green) with phalloidin-TRITC (red) counterstain. Inserts show IFE sections. Scale bars: 50 micrometers (A–F).  DNA content of keratinocytes isolated from mouse skin was used to determine proportion of cells in different phases of the cell cycle. % cells in S+G2/M phase was calculated for mice treated as indicated. Cyclop: cyclopamine. Data shown are for two mice of each founder line per treatment.(5.85 MB TIF)Click here for additional data file.Figure S4Lack of VDR impairs beta-catenin induced hair follicle differentiation but not proliferation. D2 mice were treated with 4OHT for 21 days and tail epidermis was analyzed.  Double immunostaining of tail skin sections with antibodies to keratin 14 (red) and VDR (green) with Hoechst counterstain (blue).  Alkaline phosphatase activity (blue) with Fast Red counterstain. Dashed line in (C) indicates dermal-epidermal junction. (E–H) Whole mount staining for Ki67 with Hoescht (blue) and phalloidin (red) counterstains. (I) % cells in S+G2/M was determined by flow cytometry. Data shown are for two mice of each genotype.  Immunohistochemistry for cyclin D1 (brown). Positive staining is indicated by arrows. eHF: ectopic hair follicle; eDP: ectopic dermal papilla. Scale bars: 100 micrometers.(5.18 MB TIF)Click here for additional data file.Table S1TCF/Lef and VDR binding sites in the promoter regions of beta-catenin target genes. The 3 kb proximal promoter region of 91 genes upregulated more than 3 fold in transgenic skin of K14DeltaNbeta-cateninER (D2) mice treated with 4OHT for 7 days (0.03 MB DOC)Click here for additional data file.Table S2Enrichment of TCF/Lef and VDR binding sites in the promoter of beta-catenin target genes. To determine over-representation of motifs within the gene list in (0.04 MB DOC)Click here for additional data file.Table S3VDR and Lef1 binding sites in the promoter regions of Krt15, PADI3 and S1003A genes. For each gene the actual sequence present in the mouse promoter is shown , together with the corresponding consensus binding site and the regions in the ChIP analysis. Sequences are numbered in order, according to their relative proximity to the transcription start (number 1 being closest). DR: Direct Repeat. IP: inverted palindrome.(0.07 MB DOC)Click here for additional data file.Table S4VDR and nuclear beta-catenin expression in human skin tumours. 59 human skin tumors were stained for VDR and beta-catenin. Expression was scored as high, medium or low. In green are highlighted all tumors with elements of hair follicle differentiation; these had high levels of both VDR and nuclear beta-catenin. In red are indicated those infiltrative BCCs that had high nuclear beta-catenin and low VDR.(0.08 MB DOC)Click here for additional data file.Table S5Primers used for real time PCR(0.02 MB DOC)Click here for additional data file.


Wnt signaling in neurodegenerative disorders such as Alzheimer's Disease (AD). In fact, a relationship between amyloid-β-peptide (Aβ)-induced neurotoxicity and a decrease in the cytoplasmic levels of β-catenin has been observed. Apparently Aβ binds to the extracellular cysteine-rich domain of the Frizzled receptor (Fz) inhibiting Wnt/β-catenin signaling. Cross-talk with other signaling cascades that regulate Wnt/β-catenin signaling, including the activation of M1 muscarinic receptor and PKC, the use of Ibuprofen-ChE bi-functional compounds, PPAR α, γ agonists, nicotine and some antioxidants, results in neuroprotection against Aβ. These studies indicate that a sustained loss of Wnt signaling function may be involved in the Aβ-dependent neurodegeneration observed in Alzheimer's brain. In conclusion the activation of the Wnt signaling pathway could be proposed as a therapeutic target for the treatment of AD.Recent evidence supports a neuroprotective role for  Alzheimer's disease (AD) is a neurodegenerative disorder associated with aging and characterized by fibrillar deposits of Aβ in subcortical brain regions. Typical features of AD are extracellular neuritic amyloid plaques (senile plaques) and intracellular neurofibrillary tangles. The main proteinaceous component of the amyloid deposited in AD is the Aβ peptide, a 40-to 42-residue peptide that has been isolated from senile plaque cores. Studies in AD mouse models and AD patients support the hypothesis that Aβ causes "synaptic failure" before plaques develop and neuronal cell death occurs; such effects are produced by Aβ oligomers, which are soluble and toxic molecular forms of Aβ .Wnt (wingless-type murine-mammary-tumour virus integration site) signaling in many adult and developmental processes, such as gastrulation, axis formation, cell polarity, organ development and maintenance of stem cell pluripotency, is widely acknowledged [Wnt factors controls the organization of the body plan during the early stages of development as well as organogenesis at later developmental stages. Postnatally, Wnt signaling is involved in normal biological events such as tissue maturation and homeostasis and in several neoplastic pathologies. In the mammalian central nervous system (CNS), Wnt signal transduction is involved in neural induction and patterning in early embryogenesis; previous studies have also linked Wnt signaling to neurodegenerative disorders such as AD [Wnt function is implicated in the pathophysiology of neuronal degeneration of AD. Wnt signaling is complex; 19 mammalian Wnt genes have been cloned, and more than ten membrane receptors and a plethora of cofactors and regulators are known. Different mechanisms of Wnt signaling have also been identified. The best understood of these is the "canonical" pathway, in which β-catenin transduces the Wnt signal to the nucleus [Wnts involves an interaction with a receptor complex comprising members of the Frizzled (Fz) class of 7-transmembrane receptors and a member of the low density lipoprotein receptor 5/6 (LRP 5/6) family of single-pass membrane proteins. Wnt interaction with its receptor results in an increase in the stability of β-catenin, whose accumulation results in translocation to the nucleus where it can interact with members of the TCF/LEF class of transcription factors and therefore modulate gene expression. The stability of β-catenin is controlled by Wnt through the modulation of a large cytoplasmic protein complex comprised of the protein Axin (axis inhibition protein), APC (adenomatosis polyposis coli), CK1α , GSK-3β(glycogen synthase kinase 3 beta) and GβP/frat [Wnt signaling is regulated by a wide range of proteins, which act either intracellularly by affecting signal transduction, or extracellularly by interfering with the interaction between Wnt ligands and their membrane co-receptors [Wnt pathway have been described, such as Wise, the secreted frizzled-related protein (sFRP), the Wnt inhibitory factor 1 (Wif1), Cerberus, and the Dickkopf (Dkk) family of secreted proteins. Of the four known Dkk family members, Dkk-1 is uniquely described as a negative modulator of the canonical Wnt signaling, whereas, Dkk-2 for example may activate or inhibit the pathway depending on the cellular context. Dkk-1 is expressed at very low levels in the adult brain [Wnt [The importance of owledged ,3. In emch as AD -6. In fa nucleus . In thisGβP/frat . GSK-3β eceptors . Differelt brain , and binain [Wnt .Wnt signaling [Drosophila genes involved in Wg signaling, dally  and dlp , reveal a predicted protein sequence that resembles the protein cores of glypican (HSPG) [dally alleles exhibit some wing-margin defects, a phenotype similar to partial loss of Wg activity [Dally's sensitivity to heparin lyase II and not to chondroitinase ABC treatments indicates that it contains heparan sulfate chains [Wnt-3a modulates the effect of heparin on β-catenin levels [Wnt-3a against β-amyloid neurotoxicity (Table Little is known about the role of the heparan sulfate proteoglycans (HSPGs) in vertebrate ignaling . A compan (HSPG) -16. Flieactivity . Dally'sn levels . More imWnt proteins were classified as either canonical, such as Wnt-1 and Wnt-3a, or non-canonical, including Wnt-4, Wnt-5 and Wnt-11 [Wnt proteins. Evidence suggests that Wnt-5a, for example, may activate the canonical pathway or inhibit it, depending on the receptor involved [Wnt proteins. Two non-canonical Wnt pathways have been described to play a role in development: (i) the planar cell polarity (PCP) pathway, in which Fz acts through Jun N-terminal kinase (JNK) to regulate the cytoskeleton, and (ii) the Wnt-Ca2+ signaling pathway, in which Fz activation leads to increased intracellular Ca2+ and nuclear import of the transcription factor NFAT [Wnt pathways utilized to specify pattern formation during development, but also different mechanisms. Although the final output of the canonical and the non-canonical Wnt-Ca2+ pathways are the regulation of gene expression, the PCP pathway controls planar cell polarity by modulating the cytoskeleton [Historically, d Wnt-11 ,18,19. Tinvolved . Accorditor NFAT . These sskeleton ,23.Wnt signaling pathway activity and AD. Early studies in our laboratory suggested a relationship between Aβ-induced neurotoxicity and an impairment of this signaling pathway, Figure Wnt signaling components are altered in AD [Wnt signaling pathway may be used as a therapeutic strategy to treat AD.At the beginning of this decade (Early in 2000), we found a relationship between a loss of the ed in AD -33. As aWnt signaling may play in AD. β-catenin levels are markedly reduced in AD patients carrying presenilin-1 (PS-1) inherited mutations [Wnt signaling function could be involved in the Aβ dependent neurodegeneration observed in AD [A considerable amount of data has led to a quest to understand the role utations . In factutations -36. Earlutations ,26,37. Ted in AD ,37.Wnt canonical, has its activity related with the neuropathology present in AD. GSK-3β is widely expressed throughout the rat CNS [The enzyme GSK-3β, a key modulator in the  rat CNS , with pa rat CNS . The pre rat CNS . Previou rat CNS . In summ rat CNS .Wnt signaling function may be involved in the Aβ dependent neurodegeneration observed in AD [. Recent studies in our laboratory, using double transgenic mice (APPSWE + PSEN1ΔE9) indicated that lithium injection prevents the behavioral disturbances of the animals, reducing the size of the amyloid plaque, Figure In AD brain, active GSK-3β  is mainly found in neuronal cell bodies and neurites , where ied in AD ,27. Furted in AD ,50, and ed in AD . OngoingWnt signaling: (a) destabilization of endogenous levels of β-catenin, (b) an increase in GSK-3β activity and (c) a decrease in Wnt target gene transcription. In vitro studies have shown that the activation of the canonical Wnt signaling pathway by Wnt-3a and Wnt-7a conditioned media were able to overcome the neurotoxic consequences induced by Aβ [The exposure of rat hippocampal neurons to Aβ result in three hallmarks related with ed by Aβ ,53. Moreed by Aβ ,55.2+ pathway signals through Dvl to induce calcium influx and the activation of protein kinase C (PKC) [Wnt target genes such as engrailed and cyclin-D1. Wnt-3a and lithium mimicked PKC activation [Wnt signaling pathway by Ca2+-dependent PKC iso-forms, may be important in controlling the neurotoxic process induced by Aβ. As a result, the activation of the Wnt signaling pathway has been proposed [The Wnt/Ca C (PKC) . The ina C (PKC) ,53. PKC tivation ,53. The proposed ,52,53,57Wnt signaling cannot be just attributed to a loss of function. The reduction in the Wnt signaling can be due to a gain of function of inhibitors of the Wnt signaling. Studies by Caricasole et al (2004) [Wnt pathway contributes to the pathophysiology of AD [The loss of l 2004)  showed tl (2004) . Dkk-1 w  showed gy of AD ,24,58,59Wnt signaling is by sFRPs, which are also capable to disrupt the Wnt network signaling. Increased expression of sFRP 1, 2, 3 and 5 has been reported in Inherited Retinal Degenerations such as Retinitis Pigmentosa, which is characterized by progressive loss of photoreceptors due to apoptosis [in vitro, in fact, sFRP2 a member of the family of secreted Frizzled-related proteins [Wnt/β-catenin or Wnt/Frizzled signaling pathway, which includes routes to apoptotic activation. As discussed by Baranski et al (2000) [Wnt signaling in some circumstances: for example, sFRP2 (SARP-1) increases resistance of MCF7 breast adeno-carcinoma cells to apoptotic signals, whereas sFRP1 (SARP-2) sensitizes the same cell [Wnt pathways are substantially disrupted in the degenerative state, and that targeting of sFRPs to key areas of the neuro-retina may mediate mechanisms promoting or antagonizing cell death, similar mechanisms may also be true for neurodegenerative diseases such as AD.Another mechanism for the inhibition of the poptosis ,61. sFRPproteins , is alsoproteins . They apl (2000)  it is alame cell  via oppoWnt signaling and AD. The analysis of single-nucleotide polymorphisms show an increased risk for AD in populations with inheritance of the apo-lipoprotein E-ε4 (APOE-ε4) allele, including both sporadic and late-onset familial forms of the disease [Wnt signaling pathway in PC12 cells upon stimulation with Wnt-7a as determined by luciferase activities and nuclear β-catenin levels [Wnt pathway, its association with AD was studied. Results unveil an association between a highly conserved coding sequence LRP 6 polymorphism  and the risk to develop late-onset AD in APOE-ε4 allele carriers. Interestingly, the Val 1062 variant of LRP 6 causes a reduced activation of a β-catenin-responsive reporter gene in HEK293T/STF recombinant cells [Wnt signaling pathway may predispose people to AD.Genetic epidemiological data show a link between  disease . Recentln levels . Epidemin levels . Genome-n levels . Since Lnt cells , suggestWnt signaling activation causes the dissociation of the multiprotein complex that contains, among others, GSK-3β and β-catenin. This prevents GSK-3β from phosphorylating β-catenin [Wnt pathway are involved in the regulation of neuronal survival and homeostasis  [Accumulation of cytoplasmic inclusion bodies in many neurodegenerative diseases, including AD, might result from dysfunction of the ubiquitin-proteasome system ,69. This-catenin . Un-phos-catenin  and moveroligin) -76. Phosroligin)  indicateroligin) . These froligin) . These rWnt pathways, referred to as Wnt/β-catenin pathway [Wnt-Ca2+ pathway [Wnt ligands and the 10 Fz has not been carried out, although some combinations seem to convey a meaningful intracellular signal [Wnt-3a [Wnt-7a [Wnt signaling pathway and Fz receptors have been shown to participate in the development and maintenance of the nervous system, little is known about the expression of Fz in the mammalian brain. Through the analysis of in-situ hybridization of adult mice brains, it was found that numerous Fz receptors and Wnt ligands are expressed across the brain [Wnt ligands, may contribute to the understanding of the in vivo Wnt signaling in the adult brain. More recently, a high-throughput methodology that allows the analysis of expression of 20000 genes, revealed that the adult brain of mice expresses different components of the Wnt signaling pathway [In mammals, Fz genes have been implicated in a variety of developmental processes, including axonal outgrowth and guidance in the central nervous system ,79, the  pathway ,84, vers pathway ,85. An er signal ,87, incl [Wnt-3a , and Fz5 [Wnt-7a . Althoughe brain . Knowled pathway .Wnt signaling pathway protects hippocampal neurons against the toxicity of Alzheimer's Aβ, however, the role played by the Wnt receptors Fz has not been studied. Recently we found that Fz1 mediates the activation of the canonical Wnt/β-catenin pathway by Wnt-3a in PC12 cells. In addition, the protective effect of Wnt-3a against the toxicity of Aβ oligomers was modulated by Fz1 expression levels. Over-expression of Fz1 significantly increased cell survival induced by Wnt-3a and diminished caspase-3 activation and β-catenin degradation, these Wnt-3a effects are potentiated by over-expression of Fz1, but not Fz2, and are significantly reduced when Fz1 is knocked down by antisense oligonucleotides in PC12 cells [Wnt target genes may be involved in these events. This was confirmed by co-transfecting both Fz1 and the inactive form of β-catenin, which did not elicit protection levels similar to those shown with endogenous β-catenin. Fz1 is expressed in the adult rat hippocampus and cortex, and in cultured hippocampal neurons where Wnt-3a also protects against Aβ toxicity, an effect that was decreased by knocking-down Fz1 expression [Wnt-3a modulated by Fz1 expression suggests that the activation of the canonical Wnt signaling pathway prevents the neurotoxicity induced by the Aβ peptide and again suggest a therapeutic potential for this signaling pathway in the treatment of AD.The activation of the canonical 12 cells . Over-expression . The neuWnt receptors. Based on this homology, they investigated the interaction between Aβ and Fz, and found that Aβ binds to the Fz CRD at or in close proximity to the Wnt binding site and inhibits β-catenin accumulation, nuclear translocation and Wnt-targeted gene transcription [Wnt signaling cascade. These results indicate that the Aβ binding site in Fz is homologous to cSP and that this is a relevant target for Aβ neurotoxicity. Furthermore, they suggest that blocking the interaction of Aβ with Fz might lead to novel therapeutic approaches to prevent neuronal dysfunction in AD.The signal transduction mechanisms involved in Aβ-induced neuronal dysfunction remain to be fully understood; the identity of the protein receptor(s) involved in neuronal Aβ binding has not been identified. Studies by Ferreira and coworkers in Brazil , have idcription . InteresWnt signaling as a therapeutic target for treatment of AD led us to evaluate potential pathways that interact with the Wnt signaling:The emerging role of (a) Cholinergic dysfunction has been observed in AD patients, indicating its relationship with Aβ neurotoxicity. Degenerated pre-synaptic cholinergic neurons that ascend from the basal forebrain to cortical and hippocampal areas have been observed [in vitro was later found in vivo; chronic treatment with the specific M1 agonist AF267B, improved the spatial memory and reduced the Aβ load in the hippocampus of a triple transgenic mouse [Wnt signaling pathway interact, leading to potential neuroprotection against Aβ toxicity.observed . In relaobserved  and tau observed . In addiobserved ,98. Hippic mouse . Thus, t(b) The use of non-steroidal anti-inflammatory drugs (NSAIDs) has been observed to reduce the risk for AD [1–42 in cells in vitro [Wnt signaling activation, since the increase in the activity of GSK-3β induced by Aβ is down-regulated by co-treatment with IBU-PO. In addition, this down-regulation occurs through induction of Ser-9 phosphorylation. Transgenic mice that over-express GSK-3β show low levels of Ser-9 phosphorylation and the IBU-PO treatment induces an increase in this phosphorylation [Wnt signaling pathway, could eventually rescue neurons from cytotoxicity through GSK-3β inhibition, which may be of potential benefit for the treatment of AD patients.k for AD . The NSAin vitro -104. A bin vitro . The prorylation . Compoun(c) Treatment with some antioxidants has been suggested as an avenue for the treatment of AD [Wnt signaling pathway. Treatments with Trolox  and 17β-estradiol, but not vitamin C, increases the cytoplasmic levels of β-catenin and inhibits the increase in GSK-3β activity observed when neurons are exposed to Aβ [Wnt signaling by anti-oxidant treatment increases the mRNA levels of some of the components of the Wnt pathway. Results indicated that both Wnt-7a and Wnt-5a ligands were induced by the anti-oxidant treatment [Wnt ligands  present in embryonic hippocampal neurons are also expressed in the adult rat brain [Wnt ligands are present throughout the entire lifespan of mammals.nt of AD . We haveed to Aβ . In thisreatment . A similat brain , indicat(d) The relative importance of hydrogen peroxide and free radicals in the neurodegenerative processes triggered by Aβ had been previously addressed [Wnt signaling pathway [ddressed . For exaddressed , demonstddressed . The dru pathway .Wnt signaling components, including the inhibition of GSK-3β activity, the increase in both the cytoplasmic and nuclear levels of β-catenin, as well as the transcription of Wnt target genes en-1 and cyclin-D1 [Wnt target gene [SWE + PSEN1ΔE9 double transgenic mice, indicated that rosiglitazone administration prevents the behavioral and the inflammatory-glial disturbances observed in transgenic animals, thus reducing amyloid plaque size, Figure Another PPAR, known as the PPARγ, plays an important role in the regulation of lipid metabolism . In addiyclin-D1 . Previouget gene ,116. Recget gene . In factget gene . These rget gene . Interesget gene . Recent (e) Recently, we reported that Wnt-7a induces dissociation of the APC protein from the α-catenin cytoplasmic complex and the interaction of APC with the α7-nAChR in hippocampal neurons. In the CNS, α7-nAChRs are involved in several aspects of brain function, affecting neuronal development [Wnt-7a is able to induce the re-localization of APC to membranes, clustering of APC in neurites, and co-clustering of APC with the presynaptic protein markers, including P-synapsin, SV2, and synaptotagmin. Moreover, Wnt-7a also increases the number and size of co-clusters of α7-nAChRs and APC in pre-synaptic nerve terminals [Wnt-7a increases both nAChR α7 subunit levels in an APC independent manner and clusters of α7-nAChRs in neurites via an APC dependent process [Wnt pathway by regulating the pre-synaptic localization of APC and α7-nAChRs, with APC serving as an intermediary in the α7-nAChR re-localization process. Activation of α7-nAChR with nicotine protects culture of hippocampal neurons from Aβ aggregates and this protective effect of nicotine was blocked by α-bungarotoxin. These effects are observed at the immunofluorescence level, as well as at the level of β-catenin by western blot (Figure elopment , learninelopment . Becauseelopment . In Parkelopment ,123. Wnterminals . These s process . These rWnt signaling may be essential for α7-nAChR expression and function in synapses. Perhaps therapies aimed to activate Wnt signaling could be effective in treatment of AD, especially if they prevent loss of α7-nAChRs from synaptic regions, as well as of other important synaptic proteins. These compounds and new ones yet to be discovered, which inhibit the GSK-3β activity and/or enhance Wnt signaling, could lead to the reduction of neuropathological factors involved in AD. The crosstalk of the Wnt signaling pathway with other cellular pathways is opening new possibilities for therapy.Modulation by Wnt signaling may play a role in the pathogenesis of AD. The potential use of GSK-3β as a clinical target in AD has been discussed, including the activation of M1 muscarinic receptor and PKC, the use of anti-inflammatory-ChE bifunctional compounds, PPAR agonists, and some antioxidants, all of which may play a role by regulating the Wnt/β-catenin signaling. So far the mechanisms by which extracellular Aβ causes its different intra-neuronal effects have not been clarified. Wnt-7a signaling stimulates clustering of pre-synaptic proteins and modulates the synaptic vesicle cycle by inducing recycling and exocytosis of synaptic vesicles. Aβ oligomers bind to the central synapse at the postsynaptic region, and we have found that Wnt-5a plays an attenuating role in Aβ neurotoxicity. In addition, this ligand modulates the insertion of glutamate receptors in the postsynaptic region of synapses. All of these data opens a novel therapeutic window in AD treatment.Several lines of evidence indicate that deregulated The authors declare that they have no competing interests.NCI participated in the design of the review and writing of the manuscript. EMT performed some experiments presented, and contribute to the writing and revision of the manuscript. Both authors read and approved the final manuscript.


The E-cadherin catenin system acts as an invasion suppressor of epithelial malignancies. However, it is debatable whether expression of E-cadherin or catenins is a useful prognostic marker in invasive breast cancer.We measured the expression of E-cadherin and catenins  in human breast carcinomas using real time quantitative polymerase chain reaction (Q-PCR) and investigated whether the expression levels were associated with known tumour variables or patient survival (median follow-up 72.2 months). RNA from frozen sections of breast tissue  was reverse transcribed, quantified and analysed by Q-PCR with results expressed as number of copies of transcript/50 ng RNA.There was no statistically significant difference in the expression of E-cadherin and catenins in the 33 paired normal background and tumour tissues. The expression of E-cadherin, α-, β-, and γ-catenin in node positive tumours was similar to node-negative tumours. E-cadherin, α-, β-, and γ-catenin expression in breast tumours was not related to Nottingham Prognostic Index (NPI). There was no significant difference in the expression of E-cadherin, α-, β-, γ-catenin between the various TNM stages. None of the molecular markers significantly influenced survival. Lymph node status was the only significant predictor of survival.Using real time quantitative PCR there was no difference in the expression of E-cadherin, α-, β-, γ-catenin between tumour and normal breast tissue. Furthermore, measurement of expression of these molecules was not of prognostic value in predicting long term outcome of women with breast cancer. Development of malignant tumours is in part characterized by the ability of a tumour cell to overcome cell-cell adhesion and to invade surrounding tissue. The E-cadherin catenin complex localized to actin-based adherens junctions plays a crucial role in epithelial cell-cell adhesion and in the maintenance of tissue architecture. Perturbation in the expression or function of this complex results in loss of intercellular adhesion, with possible consequent cell transformation and tumour progression.The main structure of E-cadherin catenin complex consists of transmembrane E-cadherin, cytoplasmic proteins, called catenins , and actin cytoskeleton filament. β-catenin and γ-catenin share approximately 65% sequence homology and bind directly to the cytoplasmic tail of E-cadherin in a mutually exclusive manner. α-catenin then links the bound β- or γ-catenin to the actin microfilament network of the cytoskeleton,2.In several carcinomas including gastric, head and neck, bladder, prostate, colon, and breast, a reduced or absent expression or abnormal location of E-cadherin has been observed -8. StudiWe determined the expression of E-cadherin, α-, β- and γ-catenin in human breast carcinomas using real time quantitative polymerase chain reaction and investigated whether the expression levels are associated with known tumour variables or patient survival.Tumour tissue and normal background tissue  were collected with ethical approval and informed consent from patients with invasive breast cancer. The fresh tumour and normal background tissues were snap frozen in liquid nitrogen and stored at -70°C.An overview of the clinical and pathological characteristics is summarised in Table The levels of E-cadherin, α-, β- and γ-catenin transcripts from the prepared cDNA were determined by real time quantitative RT-PCR, based on the Amplifluor™ technology, using the method previously reported SpecificThe primary antibodies used were monoclonal anti-E-cadherin , anti-α-catenin , anti-β-catenin  and anti-γ-catenin .Immunohistochemical staining was performed on 25 matched tumour and normal background tissue pairs. Frozen sections of breast tumor and normal background tissue were cut at a thickness of 6 μm using a cryostat. The sections were mounted on Super Frost Plus microscope slides, air-dried and then fixed in a mixture of 50% acetone and 50% methanol. The sections were then placed in 'Optimax'  wash buffer for 5–10 min to rehydrate. Sections were incubated for 20 min in a 0.6% BSA blocking solution and probed with the primary antibody. Following extensive washings, sections were incubated for 30 min in the secondary biotinylated antibody . Following washings, Avidin Biotin Complex (Vector Laboratories Ltd) was then applied to the sections, followed by extensive washings. Diaminobenzidine chromogen (Vector Laboratories Ltd) was then added to the sections, which were incubated in the dark for 5 minutes. Sections were then counterstained in Mayer's haematoxylin and dehydrated in ascending grades of methanol before clearing in xylene and mounting under a coverslip.Slides were viewed using a 20 × 20 objective lens  and images were subsequently saved as a 24 bit colour JPEG file image via a digital camera , and computer  equipped with a frame grabber . The captured images of tumour and normal background tissues were amalgamated using the PHOTOMERGE option in Adobe Photoshop 6. The images were then converted into gray scale images and inverted using Adobe Photoshop 6 before analysing using Optimas image analysis software .The intensity was analysed using point morphometry. 10 representative points were marked on each image captured. Overall, optical intensity data (mean and SD) was calculated by summing up the data from all images in the two groups and subtracting the mean background reading.The data obtained was analysed using the MINITAB 13.32  programme. Statistical significance was calculated using the two-sample student t-test, non-parametric Mann-Whitney test and ANOVA where appropriate. Multivariate analysis was done for survival.The intensity of membrane staining for E-cadherin and all catenin molecules was significantly more in normal background tissues compared with tumour tissues .In contrast, no statistically significant difference was seen in the expression of E-cadherin, α-catenin, β-catenin and γ-catenin in the 33 paired normal background and tumour tissues .The expression of E-cadherin, α-, β- and γ-catenin in node positive tumours was similar to node-negative tumours .There was no significant relationship of E-cadherin, α-catenin, β-catenin and γ-catenin in breast tumours to Nottingham Prognostic Index (NPI) .There was a trend towards decreased E-cadherin expression in Grade 2 and 3 tumours compared to Grade 1 tumours but the differences were not statistically significant. α-catenin expression was significantly increased in Grade 2 tumours compared with Grade 1 tumours (p = 0.03). However, α-catenin expression in Grade 3 tumours was similar to Grade 1 tumours. β-catenin expression was similar in Grade 1 and 2 tumours. However, its expression was significantly increased in Grade 3 tumours compared to Grade 2 tumours (p = 0.054). γ-catenin expression was similar in the 3 groups.Surprisingly, there was no difference in the expression of E-cadherin catenin complex between ductal and lobular tumours.The TNM Stages 3 and 4 were combined into a single group for analyses as they were very small. There was no significant difference in the expression of E-cadherin, α-, β-, and γ-catenin between the various TNM stages .E-cadherin expression in tumours of patients who were disease free was significantly more compared to those with metastatic disease, local recurrence or dying from breast cancer  p = 0.012). There was no difference in α-catenin and γ-catenin expression between the groups. β-catenin expression was increased in patients dying from breast cancer compared to disease free patients and the difference approached statistical significance (p = 0.052). Multivariate analysis was done using SPSS for mortality. None of the molecular markers significantly influenced survival. Lymph node status was the only significant predictor of survival.In this study, we examined the expression of cell-cell adhesion molecules E-cadherin, α-, β- and γ-catenin in human breast cancer by quantitative real time polymerase chain reaction. The mRNA levels of these markers were related to clinicopathological variables and survival data.The data from this study did not show any difference in the expression of E-cahderin, α-, β- and γ-catenin between tumour and related normal tissue. This contrasts with the results of immunohistochemical staining in the present study and most previously reported studies of E-cadherin catenin complex in breast cancer, which have described down-regulation of some of these molecules in tumourigenesis,23-26,28The disparity can be easily explained as this is the first study to measure expression of the E-cadherin catenin complex molecules by quantitative real time polymerase chain reaction. Direct comparisons are not possible as previous studies have varied widely in patient samples and immunohistochemical scoring methods, which make comparisons difficult.It is possible that a defect in the E-cadherin catenin complex without a change in its expression may be responsible for the malignant progression. Immunohistochemical staining and Q-PCR reveal different information and each has its advantages and disadvantages. Immunohistochemical analysis provides vital information on the protein location in the cells, which is important when studying cell adhesion molecules. However, the method has obvious limitations in quantifying the true amount of the protein in cells or tissues. Quantitative analysis of mRNA as presented here has the unique advantage in providing quantitative information of the gene expression concerned. However, this method does not provide information about the location of the molecule within a cell and may be considered 'over-sensitive'.The expression of E-cadherin, α-, β- and γ-catenin was similar in node positive and node negative tumours. Our results suggest that expression of E-cadherin, α-, β- and γ-catenin may persist into the later stages of breast carcinoma. Siitonen et al. and Oka et al. found a correlation between loss of E-cadherin and the presence of nodal metastases,30, but In contrast to most previous IHC studies -35, mRNAE-cadherin expression was not associated with tumour grade. Reduced E-cadherin expression has been associated in the past with high histological grade,36. Our et al., and Heimann et al., reported reduced expression of E-cadherin to be associated with tumour recurrence, metastases, and poor prognosis in breast cancer[There are conflicting reports in the literature regarding the relationship between E-cadherin catenin complex and prognosis/survival. In this study, E-cadherin catenin complex expression was not prognostic in breast cancer patients or related to survival. Asgeirsson st cancer,16. Not st cancer,19,37. Ost cancer,23,26,38Despite the apparent advantages of quantitation and sensitivity, the use of quantitative real time polymerase chain reaction does present some drawbacks. The technique is highly sensitive and any contaminating cells  will lead to readings that belong to non cancer cells. To overcome this problem, it is essential to isolate pure populations of tumour cells by micro dissection.In conclusion, using real time quantitative PCR we have shown that there is no difference in the expression of E-cahderin, α-, β- and γ-catenin between tumour and normal breast tissue. Furthermore, measurement of expression of these molecules is not of prognostic value in predicting long term outcome of women with breast cancer.The authors declare that they have no competing interests.AG conducted the study, analyzed the data and prepared the manuscript, TAM contributed to the conduct of the study, REM contributed to clinical follow ups and helped in editing the manuscript, WGJ contributed to the conduct of the study, design of primers and statistical analysis.


Trivers and Willard predicted that when parental condition has differential effects on the fitness of male and female offspring, parents who are in good condition will bias investment toward the sex that benefits most from additional investment. Efforts to test predictions derived from Trivers and Willard's model have had mixed results, perhaps because most studies have relied on proxy measures of parental condition, such as dominance rank. Here, we examine the effects of female baboons condition on birth sex ratios and post-natal investment, based on visual assessments of maternal body condition. We find that local environmental conditions have significant effects on female condition, but maternal condition at conception has no consistent relationship with birth sex ratios. Mothers who are in poorer condition at the time of conception resume cycling significantly later than females who are in better condition, but the sex of their infants has no effect on the time to resumption of cycling. Thus, our findings provide strong evidence that maternal condition influences females' ability to reproduce, but females do not facultatively adjust the sex ratio of their offspring in relation to their dominance rank or current condition. When parental condition has differential effects on the fitness of male and female offspring at weaning, and variation in condition at weaning persists into adulthood, parents who are in good condition are expected to bias investment toward the sex that benefits more from additional investment Nearly all of these studies relied on proxy measures such as maternal dominance rank, parity, litter size, and food availability, that were assumed to be related to physical condition. Cameron found that the small subset of studies that relied on measures of maternal condition, such as body weight, at the time of conception produced substantially stronger support for the Trivers and Willard model: in 22 of these 25 studies, females in good condition produced substantially more sons than females in poor condition. Based on these findings, Cameron suggested that “sex-adjustment occurs at or near implantation” and concluded that the inconsistency of results in the literature may be largely due to the use of inaccurate proxies for maternal condition.Cameron's meta-analysis raises interesting questions about facultative adjustments of birth sex ratios in nonhuman primate groups. There have been more than three dozen studies of facultative birth sex ratios in a range of nonhuman primate species, and nearly all have relied on maternal dominance rank as an indirect measure of maternal condition. Some studies showed that high-ranking females produce significantly more sons than daughters, while others showed the opposite pattern, and some showed no consistent pattern at all. Two meta-analyses of somewhat different sets of these studies demonstrated that the difference in the birth sex ratios of high- and low-ranking females declined as the sample size increased Macaca sinica) in Sri Lanka, high ranking females were generally heavier than lower ranking females Macaca mulatta) on Cayo Santiago Island, middle-ranking females produced significantly more daughters than higher- or lower-ranking females did Cameron's results suggest that more consistent results might be obtained with more accurate measures of maternal condition. To date, only two studies of cercopithecine primate species have examined the effects of maternal condition rather than dominance rank to test the predictions of the Trivers and Willard model. In free ranging toque macaques  were translocated from the Rift Valley near Gilgil, Kenya (Kekopey) to the Laikipia region in 1984 Opuntia stricta, a non-indigenous cactus, has invaded the area.The study groups range in an area that is topographically diverse and averages 1718 m above sea level Acacia species. Succulents make up a significant part of the diet ; these include Sansevieria, Euphorbia, and Opuntia species. Seasonality is an important feature. Food availability generally declines in the dry season Baboons feed on a variety of grasses, herbs, sedges, and the flowers and fruits/pods of a variety of shrubs and trees including several The data comprise information about births, deaths, and female reproductive status extracted from the project records. Information about maternal age at conception was not available for the original indigenous STT females. Birth and death dates of mothers and their infants were used to compute maternal age at the time each infant was born and to determine how long each infant lived. For full term births, the date of conception was computed by subtracting 177 days from the date of birth We calculated the number of days that elapsed between the birth of an infant and the resumption of a female's estrous cycling using project records on female reproductive condition. Time to resumption of cycling may be over-estimated if cycles are missed or subsequent pregnancies are not detected. To exclude this possibility, we identified outliers using box plots; intervals following birth of surviving and non-surviving infants were considered separately. For infants that died within six months, values that exceeded 280 days were excluded (n = 2). For infants that survived six months, no outliers were detected.Information about time to resumption of cycling was unavailable for 7 infants who died before they were 100 days of age. Their mothers would have been expected to resume cycling within about 94 days see . Only twDominance ranks were assessed on a monthly basis using daily ad libitum observations of avoidance and aggression between females scored on a one/zero basis per day per category of behavior. Females were arranged into a linear dominance hierarchy by minimizing the number of reversals below the diagonal. Females are categorized as high-ranking (top third of the dominance hierarchy), middle-ranking (middle third), or low-ranking (lower third). Dominance relationships among females have remained relatively stable across time.Visual assessments of maternal condition were made four times per month using calibrated photographs to score different body parts. Observers recorded the condition of (1) ischial callosities (extent of protrusion and flaking), (2) visibility of the ribs, (3) visibility of the pelvis, (4) tail stiffness, and (5) extent and location of hair loss. Each feature was scored independently from 0 (best) to 5 (worst).Baboons' pelvis and ribs are not seen normally but both become more visible when animals experience hair loss and weight loss associated with decline in condition. In addition, water and fat are lost from the paracallosal area making the ischial callosities protrude. The callosities themselves may begin to flake as the hair that normally forms the hardened pads begins to disintegrate. Finally, baboons' tails may become stiff and inflexible.We extracted the mother's condition records during the week of conception. If no condition records were available for the week of conception, then we used the condition records for the week prior to conception. If information about the week before conception was also missing, then we checked for records two weeks before conception and one week after conception. If these two values were the same, we used these values; if the values differed, we treated this as missing information. Information was available for 78% of infants conceived during the study period.The scores for the five condition features were highly inter-correlated, and a factor analysis confirmed that all of the features loaded onto a single factor. Therefore, we computed the average value of the individual scores to obtain an aggregate measure of female condition. Low values correspond to females in good condition, and high values correspond to females in poor condition.2 using the adjusted equation HB = total hits ×0.847 Herbaceous biomass was measured monthly using the slanting pin intercept technique angled 65° from vertical The dataset includes information about 79 females who gave birth to 204 infants. Because some females gave birth to multiple infants, the data are not independent. Thus, we used Generalized Linear Mixed Models with mother's identity as a random factor. When the dependent variable was continuous  we used a linear regression form of the GLMM, and when the dependent variable was binary (infant sex) we used the logistic regression form of the GLMM.The GLMM assumes that residuals are normally distributed and homogenous. For each model that we constructed, we examined the distribution and homogeneity of residuals. Residuals were plotted against the fitted values to determine whether the distribution of the fitted values were similar along the entire range of residual values. We detected no strong evidence of deviations from normality or homogeneity.Preliminary analyses showed that biomass at conception and maternal age had nonlinear effects on maternal condition. To reduce colinearity between the linear and quadratic terms, these variables were centered on the mean. We evaluated potential interactions among predictor variables , but found no significant effects, and dropped interaction terms from the models reported here.All analyses were conducted with STATA 11.0 (StataCorp 2009). All statistical tests are two-tailed. Means and standard deviations are given, where appropriate. Sample sizes vary across analyses because information about maternal age was not available for all females.Information about condition at conception was available for 79 females, ranging in age from 5.4 to 22.3 years, who produced 204 full-term infants. Condition values for conceiving females ranged from 0 to 3, with a mean and standard deviation of 1.23±0.60.2 = 76.17, p = <0.0001, n = 176; Females' condition tracked environmental conditions and age, but not their dominance rank  or from the distribution of males and females in the full data set . The birth sex ratios of high-ranking, middle-ranking, and low-ranking females in this sample did not differ significantly from unity . Moreover, the proportion of males born to females in different rank categories did not differ .2 = 3.03, p = 0.2202, n = 202; There was no significant relationship between sex of infant and maternal condition or maternal dominance rank .2 = 127.58, p = <0.0001, n = 176). Infant sex did not have a consistent impact on the time to resumption of cycling (We considered the effects of infant sex, maternal condition at conception, and maternal rank on the time to resumption of cycling (Wald χ cycling . However cycling . In addi cycling .There have now been three studies of facultative adjustment of birth sex ratios in cercopithecine primate species that were based on measures of female condition rather than dominance rank . None of these studies have found that mothers of males are in significantly better condition at conception than mothers of females. It is possible that with larger samples that generate greater statistical power, or more accurate measures of maternal condition, more consistent patterns would emerge. However, current evidence indicates that cercopithecine primates represent an exception to Cameron's It seems possible that facultative adjustment of birth sex ratios has evolved in some mammalian taxa, but not others. Nearly half of the samples that showed a significant relationship between maternal condition at conception and offspring sex in Cameron's metaanalysis were derived from studies of rodents [from Appendix A]. Six additional studies were conducted on ungulates, four on marsupials, two on pinnipeds, and two on primates. The studies of rodents and ungulates consistently supported the prediction that females in good condition will produce more sons than females in poor condition, but the patterns were less consistent for marsupials, pinnipeds, and primates.It is possible that variation in the relationship between maternal condition and offspring sex across taxa is linked to differences in life history strategies. Condition at conception may have a more consistent impact on investment capacity in capital breeders, which use stored energy to sustain their offspring, than income breeders, which use energy that they obtain during gestation and lactation to nourish their offspring Although Cameron reasoned that maternal condition provided a more accurate measure of mothers' ability to invest in offspring than maternal rank, Sheldon and West a priori reason that these mechanisms would be effective in rodents and ungulates, but not in primates.It is possible that primates lack the physiological capacity to manipulate birth sex ratios. This is difficult to evaluate because the proximate mechanisms that regulate facultative adjustment of birth sex ratios have not been identified. A number of candidates have been suggested, including maternal glucose levels It is also possible that baboon mothers have less impact on their offspring's condition at weaning and in adulthood than ungulate and rodent mothers do. In red deer, for example, mothers' condition influences sons' condition at weaning and in adulthood Mothers might have more control over the condition of their daughters than their sons. Daughters acquire their mothers' dominance rank and remain in their natal groups throughout their lives. Although maternal rank did not influence females' condition at the time of conception, high ranking females that produced surviving infants resumed cycling significantly earlier than lower ranking females did. Similar patterns have been documented among the Amboseli baboons The Trivers and Willard model relies on the assumption that one sex benefits more from additional investment than the other, and that offspring condition at the end of the period of maternal investment is correlated with offspring condition and competitive ability in adulthood. For animals, like baboons, that mature slowly, or breed throughout the year and live in seasonal and variable environments, the second assumption may be particularly difficult to fulfill. If this is the case, then selection may not favor facultative adjustment of birth sex ratios in relation to maternal condition. More research is needed to determine whether baboon mothers' condition influences the condition of their offspring at weaning, and whether variation in condition at weaning extends into adulthood.