Microfabrication of dissolvable, swellable, and biodegradable polymeric microneedle arrays (MNs) were extensively investigated based in a nano sensitive fabrication style known as micromilling that is then combined with conventional micromolding technique. The aim of this study was to describe the polymer selection, and optimize formulation compounding parameters for various polymeric MNs. Inverse replication of micromilled master MNs reproduced with polydimethylsiloxane (PDMS), where solid out of plane polymeric MNs were subsequently assembled, and physicochemically characterized. Dissolvable, swellable, and biodegradable MNs were constructed to depth of less than 1 mm with an aspect ratio of 3.6, and 1/2 mm of both inter needle tip and base spacing. Micromolding step also enabled to replicate the MNs very precisely and accurate. Polymeric microneedles (MN) precision was ranging from ±0.18 to ±1.82% for microneedle height, ±0.45 to ±1.42% for base diameter, and ±0.22 to ±0.95% for interbase spacing. Although dissolvable sodium alginate MN showed less physical robustness than biodegradable polylactic-co-glycolic acid MN, their thermogravimetric analysis is of promise for constructing these polymeric types of matrix devices. Delivering pharmacologically potent molecules to the deeper layers of the skin, in minimally invasive manner, would serve the purpose of effective delivery; pain free, bio safe, patient friendly, self-applicable systems Transdermal minimally invasive manner means fusion of non-noninvasive  and invasive systems (hypodermic needle injections), is aimed combining advantages of both systems, and excluding their drawbacks. A great example of this manner is microneedle arrays (MNs) MNs are novel miniaturized devices, have collections of needles less than 2 mm height on assemble. MNs enable delivery of small, hydrophilic drugs, and also transportation of lipophilic, and macro molecular weight biotherapeutics through microconduits that were physically created by microneedle (MN) while disruption of the stratum corneum (SC), and do not trigger the pain receptors and blood vessels There is exhaustive research relating microneedle fabrication techniques Since there is a tremendously growing market for highly accurate micro devices as minimally invasive needles, and miniature molds; microfabricating three-dimensional microstructures is a key factor. For instance, due to being economical, fast, micromachining seems to be the promising method for creating microstructures on various materials Selecting microneedle type and application approach is of great importance. Due to being economical, bio safe, having advanced functionalized features , not causing cross contamination, possessing straightforward machinability, and presenting highly accurately repetition in large scale production, polymer materials are sought after drug matrixes, micro fluidic devices, and Lab-on-a-chip systems in biotechnology and medicine Dissolvable MNs are promising candidates for facilitating the rapid release of macromolecules. They should have significant robustness for pushing them to the skin, and their fabrication procedure should be organic free solvent, and set at ambient conditions. This strategy ensures that drugs are delivered to specific targets and taken up immediately, which is plausible for short-term applications Swellable materials can be formed by chemically or physically cross-linking polymers. Chemical cross-linking leaves traces of toxic cross linkers, thus limiting its use Biodegradable polymers require high temperatures during microfabrication of drug loaded MNs, however, they may damage the incorporated biomolecules. They could be preferred for slow release of active pharmaceutical ingredients (APIs), and heat resistive APIs In this study, dissolvable sodium alginate (SA) and hydroxypropyl cellulose (HPC) (H and M grades), swellable physically cross-linked polyvinyl alcohol (PVA) and gelatin hydrogels, biodegradable chitosan and PLGA polymers were used to fabricate solid, out-of-plane, polymeric MNs from pyramidal male master templates of the following dimensions: 900-ìm height (700-ìm column height and 200-ìm pyramid tip height), 250-ìm needle base width, 500-ìm interneedle base spacing, and 100-ìm apex diameter Almost all polymeric MN fabrication processes were combined with conventional Polydimethylsiloxane (PDMS) micromolding, due to having unique features. PDMS ) has favThis study utilized a vibration minimized micromachined microdevice with conventional micromolding technology, using the fabrication method and methodology defined for the first time in This part was carried similar to PDMS-filled ABS containers were cured overnight in an oven  at 80°C, followed by cooling to ambient temperature. The cured PDMS molds  with invOlympus BX-51 fluorescent microscope  with image processing and analysis software  was used for physical characterization of polymeric MNs. The captured image sizes visualized with 640×480 at 10× magnification. The viewfinder  mode was set to brightfield light. Interactive geometric measurements were conducted in cases where the scale magnification, scale unit, and yield ratio was 10×, micrometers, 1, respectively.Not only PDMS mold production but also fabrication of water-soluble MNs were followed from To prepare aqueous blends of the sodium alginate (SA) solution, the required amount of Fluka alginic acid sodium salt derived from brown algae was gently sprinkled over cold ultrapure Milli-Q water. The suspension was mixed with a spatula until it formed a homogenous mixture, and 90 mg of 10% (w/w) SA gel was injected into the PDMS micromold according to the above procedure.During water soluble HPC gel preparation, the rate of polymer hydration was accelerated by adding dry powder to eight times its weight of water at a temperature of less than 38°C for 2 to 6 hours (according to the viscosity) with stirring. The rate of agitation was reduced when working with high-viscosity polymers to avoid foaming of the mixture. Once a clear, lump-free mixture was formed, distilled water was used to adjust the final concentration.Excessive bubble formation was observed in cellulose-based gels such as 10% (w/w) medium viscosity hydroxypropylcellulose (HPC-M) and 5% (w/w) high viscosity hydroxypropylcellulose (HPC-H), and HPMC 100 000. Entrapped air was removed by transferring them to 50-ml conical tubes and centrifuging at 3500 rpm for 5 minutes. However, HPMC MNs were not further studied because they caused skin irritation.To prepare the PVA-gelatin MN arrays, PVA and gelatin hydrogels were physically cross-linked by cryogelation. A total of 90 mg of the cross-linked mixture (20% w/v PVA and 10% w/v gelatin) was injected into the PDMS micromold as explained in the procedure of microfabrication of dissolvable MNs, but this time, the loaded micromolds were frozen at −20°C for 12 hours, followed by thawing at 25°C for 12 hours. This freeze-thaw cycle was repeated three times.To prepare the chitosan (medium molecular weight in the range of 190 000–300 000 with degree of deacetylation ≥75%)  MN arrays, the required amount of chitosan were gradually added to half of the desired amount of glacial acetic acid. The remaining glacial acetic acid was added to yield a clear, lump-free solution with final concentration of 3% (w/w) chitosan that was then micromolded as in the above-mentioned procedure.PLGA MNs were fabricated according to a previously published paper −5 Torr and 15 kV . The specimens were first sputter-coated with an ion beam-based system  that contained a single vacuum, and the layer thickness was controlled with a film thickness monitor . Computer software  was used to display the SEM images. The magnification, tilt degree, spots, width and other imaging characteristics were reported on the SEM images.MN arrays were mounted on circular discs and morphologically characterized with an environmental scanning electron microscope (ESEM) in high-vacuum mode using the ETD detector at 10The glass to rubber transition of MN base materials, melting peak, and delta Cp were investigated using differential scanning calorimetry (DSC) system .The sealed polymer film samples in 25-μl aluminum crucibles  were heated at a linear heating rate of 10°C/min from 20 to 300°C, and nitrogen flow rates was 40 ml/min (purge gas MFC). Extrapolated onset, peak, and offset temperatures were calculated with Netzsch-compatible software .Some of the polymeric MNs were tested for bending strength using a micromechanical test machine .Axial fracture forces  and tran2) was fixed on the upper station, and MNs was mounted on the lower station of the micromechanical tester . A single MN was pressed into the metal mill at 500 µm/sec in each test  and fixed on the lower station, and a custom-made cuboidal metallic mill  was attached to the upper station of the micromechanical tester. The MN array was carefully aligned with the aid of a microscope camera  such that the metal mill could freely touch and fracture the single MN at a known height, where the test speed was 100 µm/sec . MNs were vertically attached to a square ABS plastic stub , and Master micro array templates can be fabricated by dry, wet anisotropic, X-ray, UV, and ion beam etching, excimer laser micromachining, laser drilling, hot embossing, and microinjection molding. However, most of these micro fabrication processes are slow and limited to few materials, and necessitate intricate course of actions Due to having low surface energy and abovementioned unique properties of PDMS, conventional micromolding was perfectly matched with micro milled pyramidal master needles for inverse replication of them. None of the six PDMS micromolds adhered to master templates. Theoretical measurements of the micromolds and actual measurements of the polymeric MNs were very close even after many uses of PDMS micromolds .3 (methyl) changes features of silicone, creates abovementioned unique physicochemical properties of PDMS  showed significant differences between different polymers with one way ANOVA (p<0.0001), which may have resulted from shape transitions during solidification, having different viscosities of distinct material composition.Digital photographs of MN sections from 10×10 SA , HPC-M , HPC-H , cross-lRatios of standard deviation to mean percentage of visualized needles were used to calculate the fabrication precision of polymeric MNs height, and inter base spacing using the PLGA MNs could be fabricated within ±0.18% of the desired height. The fabrication precision of the base diameter was ±0.45%, and the precision of interbase spacing was ±0.22%. Other inversely fabricated polymeric MNs precision was found as excellent as the ones reported. In order to be concise we did not report the rest of the polymeric needles  replication precision, but could be calculated from the same way.The precision of PLGA MN height was within ±0.18%, while it was within ±1.82% and ±0.86% for chitosan and SA MNs, respectively. Thus, PLGA MNs were better in terms of manufacturability, reliability, and repeatability. Another interesting finding was that the weakest material (chitosan) had a greater number of bent tips than the strongest material (biodegradable PLGA). In terms of height, PLGA MNs were 10 and 5 times more precise than chitosan MNs and SA MNs, respectively.As it was declared in The structural morphology of the polymer SA, HPC-M, HPC-H, PVA-gelatin, and chitosan MNs was analyzed  bases, an exothermic peak was observed at 44.3°C that represented crystallization of the amorphous material. Melting occurred at approximately 197.3°C, and recrystallization of amorphous material and crystals was observed at 230.4°C. The glass transition (Tg mid) occurred at approximately 139.25±0.04°C, with a delta Cp of 2.18±0.27 J/(g·K).An exothermic peak was observed at 44.5°C that corresponded to the crystallization of the amorphous HPC-M MN base material. Melting occurred between 200 and 250°C. The recrystallization of amorphous material and crystals occurred between 150 and 200°C. The glass transition (Tg mid) occurred at approximately 211.87±1.46°C, with a delta Cp of 1.92±1.32 J/(g·K).The cross-linked PVA-gelatin MN base exhibited an exothermic peak corresponding to pure crystals at approximately 44.3°C. The first melting point was observed at approximately 176.7°C, and a second melting point was observed above 200°C. The glass transition (Tg mid) occurred at approximately 122.93±8.14°C, with a delta Cp of 0.94±0.45 J/(g·K).The chitosan MN base had an exothermic peak at approximately 45.6°C that resulted from the crystallization of amorphous material. Melting occurred at approximately 221.4°C. The glass transition (Tg mid) occurred at approximately 157.97±17.51°C, with a delta Cp of 7.34±6.26 J/(g·K).p 0.0205, p<0.05).Having Tg value over room temperature increased potentiality of the SA, HPC-M, PVA-gelatin, and chitosan polymer as to be convenient material for MN preparations. Glass transition mid values for SA, HPC-M, PVA-Gelatin, and Chitosan MN bases were found significant different, when non parametric Kruskal-Wallis statistic was performed  and base (200 µm) diameters The safety margin, or fraction of the failure force to insertion force, decreased from 3.8 to 1.7 as the increasing of needle length for biodegradable PLGA MNs MNs often cannot be fully inserted into the skin because the non-uniform surface of the skin causes imprecise axial insertion that result in transverse bending of the needles The transverse failure forces of PGA needles, with 100- and 200-µm base diameters with a constant tip diameter (25 µm) and length (1 mm), were 0.058±0.012 and 0.24±0.05 N, respectively 0.1 N/needle, and 0.5 N/needle were the needle failure points for  conical CMC MNs, and PLA MNs, respectively. Pyramidal MNs made of different material compositions were rated for their robustness starting with the strongest one as, PLA, amylopectin, CMC/BSA (80∶20%), BSA, and CMC MNs. More interestingly, the combination of BSA and CMC exhibited a more robust microstructure than CMC alone Young's modulus and hardness of non-toxic Gantrez® AN-139 MN were noted as 6.56±0.56 GPa, 385.6±12.00 MPa, respectively, which indicates it as a stiffness microneedle base material SA MN exhibited elastic deformation, as evident from the force versus displacement curve, which was linear for the axial compression test (force range between 0.0 and 0.18 N) . SimilarThe micromilling and PDMS micromolding were effective methods to microfabricate solid, out-of-plane pyramidal needles high precisely and accurately with no limit to the substrate materials.For the first time, pyramidal SA, chitosan, HPC-M, HPC-H, and cross-linked PVA-gelatin MNs polymers were inversely replicated with precisions ranging from ±0.18 to ±1.82% for height, ±0.45 to ±1.42% for base diameter, and ±0.22 to ±0.95% for interbase spacing Although biodegradable polymeric PLGA MNs have more resistance and mechanical stability, dissolvable SA MNs are proved to create microholes in the skin layers without breaking


We hypothesized that Wnt/β-catenin signaling transcriptionally activates microRNA-181s in HCC.Hepatocellular carcinoma (HCC) is a malignant cancer with an observable heterogeneity and microRNAs are functionally associated with the tumorigenesis of HCC. We recently identified that EpCAM (CD326)-positive cells isolated from alpha-fetoprotein (AFP)-positive HCC samples are hepatic cancer stem cells (HepCSCs). EpCAMin vivo using chromatin immunoprecipitation assay.Using both western blot and quantitative reverse transcriptase-PCR analyses, we found that the expression of all four microRNA-181 family members was positively correlated with β-catenin expression in HCC cell lines. MicroRNA-181 expression could be directly induced upon an activation of Wnt/β-catenin signaling, which includes Wnt10B overexpression, inhibition of GSK3β signaling by LiCl, or forced expression of β-catenin/Tcf4. Moreover, microRNA-181 expression was inhibited upon an inactivation of Wnt/β-catenin signaling by an induction of adenomatosis polyposis coli (APC) expression or silencing β-catenin via RNA interference. In addition, seven putative β-catenin/Tcf4 binding sites were identified in the promoter region of the microRNA-181a-2 and microRNA-181b-2 transcripts. Consistently, we found that Tcf4 interacted with these regions Taken together, our results demonstrate that microRNA-181s are transcriptionally activated by the Wnt/beta-catenin signaling pathway in HCC. Hepatocellular carcinoma (HCC) is the fifth most common malignant cancer and the third leading cause of cancer death worldwide with the observable heterogeneity in its morphology, clinical behaviour, and molecular profiles . Studies+AFP+ HepCSCs ) and two hepatic transcriptional regulators of differentiation, i.e., caudal type homeobox transcription factor 2 (CDX2) and GATA binding protein 6 (GATA6). We also found that inhibition of miR-181s leads to a reduction in quantity and tumor initiating ability of EpCAM+AFP+ HepCSCs, suggesting the potential utility of these miRNAs as molecular targets of HepCSC [MicroRNAs  are a class of small, non-coding, endogenous and functional RNAs. Mature miRNAs are generated from sequential processing of primary miRNA transcripts by Drosha and Dicer, then serve as posttranscriptional regulators of gene expression in certain biological events including carcinogenesis . Interes HepCSCs ,10. In af HepCSC ,10. The Klotho mice where loss of Klotho, a secreted Wnt antagonist, leads to β-catenin activation [Klotho mice were higher than those of wild-type mice  . Compare2), but not in control HT29-GAL cells  can promote β-catenin degradation . To inves figure . Furthers figure  and 3C ss figure . These dStudies have shown that the β-catenin/Tcf complex transcriptionally regulates their target gene expression through binding to a consensus core TCF/LEF-binding site (5'-A/T A/T CAAAG-3') within the promoter regions ,16. GenB+AFP+ HCCs (named as HpSC-HCCs) with poor prognosis, high metastasis, and the presence of a hepatic stem cell (HpSC) feature, and EpCAM-AFP- HCCs (named as MH-HCCs) with good prognosis and the presence of a mature hepatocytes (MH) feature. We have experimentally demonstrated that EpCAM+AFP+ HCC cells are HepCSCs [HCC is an aggressive cancer with extensive clinical and genomic heterogeneity. We have previously utilized a combination of EpCAM and AFP expression to identify two distinct HCC subgroups, i.e., EpCAM HepCSCs ,5,15. In HepCSCs . These rin vivo binding between the Tcf4/β-catenin complex and the TCF/LEF binding elements in the promoter region of miR-181a-2/miR-181b-2 transcript.Our current study has demonstrated that the Wnt/β-catenin signaling pathway can directly regulate miR-181s expression in HCC. This conclusion is based on the following observations: 1) miR-181 expression was positively associated with β-catenin level in both HCC cell lines and mouse livers where β-catenin is augmented; 2) activation of Wnt/β-catenin signaling directly induced miR-181 expression, while inhibition of Wnt pathway directly reduced miR-181 expression; 3) we found evidence of + HepCSCs [Wnt/β-catenin is a key molecular signaling regulator in embryonic stem cells and in a variety of human cancers including HCC. Similarly, Wnt/β-catenin signaling is known to be involved in liver development and regeneration, and has been shown to be activated in EpCAM HepCSCs ,17-19. I HepCSCs . Our finThe Wnt/beta-catenin signaling is a major transcriptional regulator of miR-181s expression.Constitutively active β-catenin expression vector (pCI-NEO-βcateninXL) (mutated at S33Y), wild-type Tcf4 expression vector (pcDNA/myc-hTcf4) and dominant negative Tcf4 expression vector (pcDNA/myc-ΔN-hTcf4) were generous gifts from Dr. Bert Vogelstein . β-catenKlotho knock out mice were kindly given by Dr. Toren Finkel. Liver tissues were dissected from these mice and immediately stored at -80°C until RNA extraction. Transfection of siRNAs and plasmids was done using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instrument. A total of 100nM siRNAs or 2 μg/ml plasmids were used for each transfection. Total RNA was extracted three days after transfection.Human HCC cell lines  were cultured as previously described . HT29-APTotal RNA was extracted using Trizol (Invitrogen). Reverse transcription was done using High Capacity cDNA Reverse Transcription Kit for mRNAs and TaqMan MicroRNA Reverse Transcription Kit for miRNAs. The expression of mRNAs and mature miRNAs was measured using Taqman Gene Assay specific for CTNNB1 and Taqman MicroRNA Assays specific for miR-181a, miR-181b, miR-181c and miR-181d (Applied biosystems). The experiments were performed in triplicate. The Taqman gene assay for 18S and Taqman MicroRNA Assay for U6 RNA were used to normalize the relative abundance of mRNA and miRNA, respectively.Total protein was extracted as previously described . They weChIP assay was done using the Chromatin Immunoprecipitation Assay Kit (Upstate), Hep3B cells and anti-Tcf4 antibody according to the manufactures' instrument. The oligonucleotides flanking transcription start site and predicted Tcf4 binding sites were designed within ~200-bp windows in the 700-bp upstream of the transcription start site (amplicon U), and in the 500-bp windows downstream of transcription start site (amplicon D) for detecting miR-181a-2 and miR-181b-2 promoter. The primers were 5'-aaa caa agc aac tgc cat gta ct-3' (sense) and 5'-agg gtc ctt ggg gtt tat ttt-3' (antisense) for amplicon U, and 5'-ggt tgc ttt cca ttc ttt gc-3' (sense) and 5'-ttt ggc cac aca aaa cta gag at-3' (antisense) for amplicon D. The PCR was done using Platinum Taq Polymerase High Fidelity (Invitrogen).Student's t-test (two-tailed) was used for statistical analysis of comparative data between different groups. p < 0.05 was considered to be statistically significant. Data were presented as the mean ± standard deviation (S.D.) and the analyses were performed using GraphPad Prism software 5.0 .The authors declare that they have no competing interests.JJ contributed to experiment design, performing experiments, data analysis and manuscript preparation. TY contributed to the data analysis and XWW contributed to experimental design, data analysis and manuscript preparation. All authors have read and approved the final manuscript.


We propose that suppression of the canonical Wnt signaling pathway, and accompanying reduced expression of β catenin and integrin β4 in laminar basal epithelial cells reduces cell:cell and cell:basement membrane attachment, thus, destabilizing the laminar epidermal:dermal junction.The digital laminae is a two layer tissue that attaches the distal phalanx to the inner hoof wall, thus suspending the horse's axial skeleton in the hoof capsule. This tissue fails at the epidermal:dermal junction in laminitic horses, causing crippling disease. Basal epithelial cells line the laminar epidermal:dermal junction, undergo physiological change in laminitic horses, and lose versican gene expression. Versican gene expression is purportedly under control of the canonical Wnt signaling pathway and is a trigger for mesenchymal-to-epithelial transition; thus, its repression in laminar epithelial cells of laminitic horses may be associated with suppression of the canonical Wnt signaling pathway and loss of the epithelial cell phenotype. In support of the former contention, we show, using laminae from healthy horses and horses with carbohydrate overload-induced laminitis, quantitative real-time polymerase chain reaction, Western blotting after sodium dodecylsulfate polyacrylamide gel electrophoresis, and immunofluorescent tissue staining, that positive and negative regulatory components of the canonical Wnt signaling pathway are expressed in laminar basal epithelial cells of healthy horses. Furthermore, expression of positive regulators is suppressed and negative regulators elevated in laminae of laminitic compared to healthy horses. We also show that versican gene expression in the epithelial cells correlates positively with that of β-catenin and T-cell Factor 4, consistent with regulation by the canonical Wnt signaling pathway. In addition, gene and protein expression of β-catenin correlates positively with that of integrin β4 and both are strongly suppressed in laminar basal epithelial cells of laminitic horses, which remain E-cadherin The equine digital laminae span between the surface of the distal phalanx and the inner hoof wall, suspending the horse's axial skeleton and attached structures within the hoof capsule. Laminae are composed of 500 to 600 vertical folds of keratinized epidermal tissue, the primary epidermal laminae, which are contiguous with the inner hoof wall, and interdigitated folds of connective tissue, the primary dermal laminae, which are contiguous with the distal phalanx . Each foThe laminar epidermal:dermal junction is compromised in a variety of disease conditions including septic processes, systemic toxicity, metabolic or endocrinopathic deregulation, traumatic injury and sustained elevated load resulting from contralateral limb injury Failure of the laminar epidermal:dermal junction in horses with starch-gruel induced laminitis, which is a model for natural disease Wingless in Drosophila and integration 1 in mouse breast tumors, which is a glycoprotein that activates the pathway, (ii) its receptor, frizzled (FZD), (iii) its co-receptor, low density lipoprotein receptor related protein (LRP); (iv) disheveled (Dsh) which together with the Wnt/FZD/LRP complex forms a signalosome that inhibits glycogen synthase kinase 3β (GSK3β), thus stabilizing β-catenin by preventing its phosphorylation and therefore its ubiquitination and proteasomal degradation, (v) protein phosphatase 1 (PP1) and protein kinase B (Akt), which also prevent β-catenin phosphorylation, and (vi) TCF which in complex with transcriptional co-activator β-catenin induces target gene expression. Pathway components that diminish expression of β-catenin and elevate its degradation are called “negative regulators”. These are: (i) Dickkopf (DKK), which antagonizes Wnt signaling by binding the co-receptor LRP, (ii) components of the β-catenin degradation complex, namely, the scaffolding proteins Axin and adenomatous polyposis coli gene product (APC) which bind β-catenin, and (iii) casein kinase 1α (CK1α) and GSK3β which jointly phosphorylate bound β-catenin leading to its subsequent ubiquitination and proteasomal degradation.The canonical Wnt signaling pathway is one of the fundamental pathways that control cell development, proliferation, differentiation, polarity and motility. The core activity of the pathway is regulation of the production and stability of β-catenin in the cell. This is important because β-catenin mediates canonical Wnt signaling by binding to and activating members of the T cell factor (TCF) transcriptional factor family. The canonical Wnt signaling pathway regulates expression of genes encoding versican Here we determine: i) whether suppressed versican gene and protein expression which occurs in LBEC of laminitic horses This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The Protocol was approved by the Animal Care and Use Committee of the University of Missouri, College of Veterinary Medicine, Protocol # 4329 and of the Louisiana State School of Veterinary Medicine, Protocol # 11056.Healthy horses  with no previous history or signs of laminitis were used. Prior to inducing experimental laminitis all horses were subjected to a complete physical examination  and gait evaluation . At the University of Missouri College of Veterinary Medicine, horses were administered carbohydrate gruel (85% cornstarch and 15% wood flour [17.6 g/kg body weight]) by nasogastric tube, or similarly administered 6 liters of deionized water as previously described RNA was isolated from three separate sections of front hoof dorsal laminae from each horse using Stratagene Absolutely RNA kit  according to manufacturer's instructions. Briefly, flash frozen tissue was pulverized in a dry ice-chilled slammer  and homogenized in the lysis buffer provided in the kit, then processed accordingly. The purity and concentration of RNA were determined by NanoDrop 1000  and consistently yielded ratios of A260:A280 and A260:A230 close to 2.0. Integrity of isolated RNA was confirmed by electrophoresis on a 1.0% agarose gel and staining with SYBRSafe DNA Gel Stain .cDNA was synthesized using Quanta qScript cDNA Synthesis Kit . Primer sets are listed in 0.35 g snap frozen tissue was pulverized in a dry ice-chilled slammer  immediately homogenized in 10 ml of extraction buffer  on ice, extracted overnight at 4°C and protein concentration determined by Bradford Assay .An aliquot (30 µg protein content) of laminar extract from each horse was boiled in reducing (5 mM 2-mercaptoethanol) Laemmli sample buffer  for 5 min and subjected to SDS-PAGE in a 4% (w/v) polyacrylamide stacking gel with a 10% (w/v) polyacrylamide gel as previously described 10 µm frozen sections were cut from Tissue-Tek O.C.T-embedded tissues and affixed to Fisher Superfrost Plus glass slides . Indirect immunofluorescence staining was performed using a combination of antibodies that separately stain integrin β4, laminin, β-catenin, E-cadherin, Wnt4, FZD4, and vimentin  togetherStatistical analyses of gene expression and Western blot data were performed using Graph Pad Prism Version 5 . All gene expression data were normally distributed as determined by Column Statistics using the D'Agostino and Pearson Omnibus Normality Test. Protein expression data of integrin β4, β-catenin, vimentin, E-cadherin and serine-9-phospho-GSK3β, were also normally distributed. GSK3β protein expression data were not normally distributed. Normally distributed data were analyzed by one-way ANOVA with Dunnett's post hoc test, and by Pearson product moment correlation co-efficient analyses with two-tailed 95% confidence. Data that were not normally distributed were analyzed using the Kruskal–Wallis one-way ANOVA with Dunn's multiple comparisons test. In all tests a P value of <0.05 was considered significant.In the laminae of both healthy horses and of horses with starch gruel-induced laminitis, expression of the hemidesmosome component integrin β4 is restricted to the single layer of basal epithelial cells of the secondary epidermal laminae . In addiReduced expression of integrin β4 in laminitic laminae is accompanied by reduced gene and protein expression of the adherens junction and canonical Wnt signaling pathway component β-catenin , panel BGene (at OG1) and protein (at OG3) expression of the type III intermediate filament protein vimentin, which is a marker of mesenchymal cells Levels of β-catenin protein expression are regulated by the canonical Wnt signaling pathway. Levels of β-catenin gene expression may also be regulated, directly and indirectly, by the canonical Wnt signaling pathway Whereas expression of genes encoding positive regulators of the canonical Wnt signaling pathway is suppressed in the laminae of laminitic horses , expressFinally, expression of the gene encoding TCF4, which in complex with transcriptional co-activator β-catenin induces target gene expression in the canonical Wnt signaling pathway A strong positive correlation was also observed between the gene encoding TCF4 and that encoding versican , and a moderate positive correlation between the gene encoding β-catenin and that encoding versican . In addition, there was a strong positive correlation between expression of the gene encoding TCF4 and that encoding integrin β4 , and between the gene encoding and β-catenin and integrin β4 .Studies reported above show that genes and proteins encoding multiple positive regulators of the Canonical Wnt Signaling Pathway were decreased in the laminae of horses affected by laminitis. In addition, many of the known negative regulators of the wnt pathway were increased in the same conditions, suggesting that the wnt signaling pathway is negatively regulated during the progression of laminitis. This possibility is further supported by evidence that versican, one of the known targets of the Wnt pathway Versican gene and protein expression is associated with mesenchymal-to-epithelial cell transition in transfected NIH 3T3 cells Although vimentin gene and protein were not expressed in laminar basal epithelial cells of healthy or laminitic horses, vimentin was strongly expressed by connective tissue cells in the dermal laminae and expression was elevated in laminitic horses. Vimentin is a type III intermediate filament protein present in mesenchymal cells. In addition to expression by cells undergoing epithelial to mesenchymal transition, it is constitutively expressed by resting fibroblasts and expression is elevated in stimulated Despite the absence of epithelial-to-mesenchymal transition in the laminar basal epithelial cells of laminitic horses, there was evidence of impaired function. The diminished presence of positive regulators and elevated presence of negative regulators of the canonical Wnt signaling pathway in the laminar basal epithelial cells of laminitic horses correlated with diminished expression of β-catenin and integrin β4, which are components of adherens junctions and hemidesmosomes respectively Although the expression of genes encoding β catenin and integrin β4 was strongly positively correlated in equine laminae , direct regulation of integrin β4 gene expression by the canonical Wnt signaling pathway was not demonstrated, and there is no report of this in the literature. In contrast, cross talk has been reported between the canonical Wnt signaling pathway and other signaling pathways Suppressed canonical Wnt signaling may not solely be a feature of starch gruel-induced laminitis. In support of this possibility, it has been shown that the number of hemidesmosomes per micrometer of basement membrane is decreased in ponies with insulin-induced laminitis If suppression of the canonical Wnt signaling pathway in laminar basal epithelial cells is a feature of laminitis of diverse etiology, pathway agonists may help to ameliorate laminar pathology. In this regard, expression of genes encoding the adherens junction component E-cadherin and the hemidesmosome component integrin α6 was not suppressed in horses with starch gruel-induced laminitis  and E-caFigure S1The anatomy of the hoof wall : The Figure is reproduced by permission of Dr. C. Pollitt. Sections of laminae presented in the paper are from the mid dorsal front hoof laminae and the region is delineated by the oval shown in this figure.(TIF)Click here for additional data file.Figure S2Protein expression in NP-40 extracts of healthy and laminitic equine digital laminae: Western blots of 0.5% NP-40 extracts (30 ìg protein/lane) from laminae of healthy horses , horses with OG1-lameness (n = 6) and horses with OG3-lameness (n = 5) showing expression of: A. integrin β4; B. β-catenin; C. vimentin; D. GSK 3β; E. serine-9-phospho GSK3β (pGSK 3β); F. E- cadherin; β actin was used as load control. The bands enclosed by the black box represent a common sample (from the OG1 group) that was run in all gels and used for normalizing experiment variation. The intensity of chemiluminescence was quantified and values were statistically analyzed shown in (TIF)Click here for additional data file.Figure S3Distribution of Wnt4 and FZD4 in healthy and laminitic equine digital laminae: 10 µm sections of frozen laminae from: A, D - a representative (n = 3) healthy horse (CON), B, E - a representative (n = 4) horse with OG1-lameness (OG1) and C, F - a representative (n = 3) horse with OG3-lameness (OG3) were stained red with antibodies against Wnt4  and FZD4  . Nuclei are stained blue with DAPI. Images were taken with a 20× objective. Scale bars 50 µm.(TIF)Click here for additional data file.Figure S4E-cadherin protein expression and integrin α6 gene expression:A - Protein expression of E-cadherin relative to the protein expression of β actin. B - Gene expression of integrin α6 relative to the gene expression of GapDH. Horizontal lines indicate mean ± standard error of mean of n = 8 CON, n = 6 OG1, and n = 5 OG3 animals. * = p<0.05 as calculated by one-way ANOVA.(TIF)Click here for additional data file.


To review the evidence of the involvement of the Wnt signalling pathway in mood disorders and in the action of drugs used to treat these disorders. We performed a careful PubMed search using as keywords all possible terms relevant to the Wnt pathway and crossing them with each of four areas, i.e., developmental effects, behavioural effects, mood disorders, and drugs used in their treatment. Papers were selected on the basis of their content and their data used for discussion. Neurodevelopmental and behavioural data point to the possibility of involvement of the Wnt pathway in the pathophysiology of mood disorders. Clinical and post-mortem data are not sufficient to corroborate a definite role for Wnt alterations in any mood disorder. Combining genetic and pharmacological data, we may state that glycogen synthase kinase is the key molecule in bipolar disorder, as it is connected with many other signalling pathways that were shown to be involved in mood disorders, while Wnt molecules in the hippocampus appear to be mainly involved in depressive disorders. Altered Wnt signalling may play a role in the pathophysiology of mood disorders, although not a central one. It is premature to draw conclusions regarding the possible usefulness of Wnt manipulations in the treatment of mood disorders. In adTo date, there is no evidence that abnormalities in the Wnt signalling pathway are causally related to any psychiatric disorder; however, alterations of individual components of the pathway have been found in psychiatric disorders. The evidence that β-catenin levels are reduced in the hippocampal CA3 and CA4 regions, and Wnt1 levels are increased in the hippocampal CA4 region of post-mortem schizophrenic brains ,6 foster2We searched the PubMed database using different strategies according to the section of the review. We used a general set of key words with all expressions denoting the pathway itself or the molecules known to be components of the Wnt signalling pathway . This set we used in all individual searches. To review the role of the Wnt pathway in human physiology and normal development, we crossed the above set with other key words, like neurodevelopment, development, apoptosis, cellular death, proliferation, oncogenesis, physiology, subsequently restricting to human studies. To gather behavioural studies of the Wnt pathway, we used the same strategy as above regarding the pathway, and crossed with general terms like behaviour and behavioural, specific terms like depression, psychosis, anxiety, hyperactivity, exploratory, immobility, avoidance, aggression, aggressiveness, aggressive, sedation, sniffing, grooming and the like, and paradigm-related terms, like forced swim test, tail suspension, open-field, marble burying etc. To identify studies dealing with the involvement of the Wnt pathway in mood disorders, we crossed the above set with another set containing terms like bipolar, depression, depressive, dysthymia or dysthymic, mixed state, agitation, mania, manic, rapid cycling, hyperthymic, dysphoric or dysphoria, suicide, suicidality, killed and self, and mood. Finally, to explore the effects of drugs used in the mood disorders on the Wnt pathway, we crossed the general set with subsets composed of keywords, like mood stabilisers or each one of them , antipsychotics or each antipsychotic, and antidepressants or each antidepressant. We then selected retrieved articles according to their relevance and further searched their References.3The Wnt family in humans consists of 19 secreted proteins, termed Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, and Wnt16. All these proteins undergo post-translational modifications and are 350-400 amino acid-long.Wnt ligands interact with a number of surface receptors, which include Frizzled (Fzd) receptors, the low-density lipoprotein receptor-related protein 5 and -6 (LRP5/6), and the tyrosine kinase receptors, Ryk and Ror -15.1). β-Catenin is an intracellular protein that exert pleiotropic functions, including adherent junction formation and gene expression regulation (see below).Wnt signalling pathways are classified as β-catenin-dependent and β-catenin-independent, also known as canonical and non-canonical Wnt pathways, respectively  in the N-terminus domain of the enzyme. The precise details of Wnt inhibition of GSK3β are unclear. One should bear in mind that the canonical Wnt pathway prevents the phosphorylation of β-catenin by GSK3β, but could leave other GSK3β functions intact. However, studies in Wnt overexpressing PC12 cells show that (i) activated Dvl recruits protein kinase B (Akt) at the β-catenin destruction complex; (ii) Akt inhibits β-catenin degradation by phosphorylating GSK3β on Ser9; and (iii) a negative dominant mutant of Akt decreases free β-catenin levels [In the “canonical” pathway, Wnt binds to Fzd and LRP5/6, thereby recruiting the scaffold protein, Dishevelled (Dvl), to the ligand-receptor complex. Activated Dvl binds to, and destabilises, a “β-catenin-destruction complex”, composed by GSK3β, adenomatous poliposys coli (APC), and axin. Within the destruction complex, GSK3β phosphorylates β-catenin, which is then committed to proteasomal degradation . It is nn levels .2+ pathways [1). In the Wnt/Ca2+ pathway, Wnt5a signalling through Fzd and Dvl activates phospholipase-Cβ, which stimulates the hydrolysis of phosphatidylinositol-4,5-bisphosphate with ensuing formation of the second messengers, inositol-1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG). InsP3 stimulates Ca2+ release from intracellular stores, whereas DAG facilitates the activation of protein kinase C (PKC). PKC in turn activates Cdc42 (cell division control protein 42), a regulator of cell adhesion, cell migration and tissue pattering. The increase in intracellular Ca2+ activates a mitogen-activated protein kinase (MAPK) pathway composed of the transforming growth factor-β-activated protein kinase (TAK1), which is a MAPK kinase kinase, and the nemo-like kinase (NLK), which is a MAPK. This pathway negatively regulates the canonical Wnt pathway by inhibiting gene transcription mediated by the β-catenin/TCF complex [Some Wnt isotypes, including Wnt4, Wnt5a, and Wnt11, are able to activate “non-canonical” β-catenin-independent pathways, such as the Planar Cell Polarity (PCP) and Wnt/Capathways ,22 , Sonic Hedgehog (SHH), and Bone Morphogenetic Proteins (BMPs) . Wnts, l5The pathophysiology of most psychiatric disorders is still unclear. Recent data indicate a neurodevelopmental origin for schizophrenia -38, bipo6Recent data show that GSK3 regulates mood-related behaviour. The expression levels of GSK3β are associated with depression- and anxiety-like behaviours, as shown by an increased exploratory activity , reducedFinally, GSK3 activity regulates circadian rhythms, with genetic depletion or inhibition of GSK3 resulting into a significant delay in the periodicity of the endogenous clock .77.1Bipolar disorder (BP), also known as bipolar affective disorder or manic-depressive illness, is associated with alterations in synaptic formation and plasticity . BP caus7.1.1Rats treated with the mood stabilisers, lithium and valproate, show increased levels of β-catenin, which is indicative of an activation of the Wnt pathway, in the frontal cortex . GSK3β i7.1.2et al., 1997), and this reduction is associated with glial cell loss and reduced neuronal number or size [Post-mortem studies suggest that defects in neurodevelopment and neuroplasticity underlie BP. Grey matter volume is reduced by about 40% in the subgenual prefrontal cortex of BP patients   have not been associated with BP .7.2As in bipolar disorder, abnormalities in neuronal plasticity and neurogenesis strongly concur to the pathogenesis of MDD. Recent studies suggest an important role for Wnt signalling pathway in MDD.Different Wnt isotypes, including Wnt2, 3a, and 7a, are expressed in the rat hippocampus, where an increased Wnt2 expression was shown after chronic treatment with various classes of antidepressants  or afterAlong the Wnt signalling pathway, various components have been specifically implicated in physiological neurogenesis and its anomalies, like Frizzled receptors, Dvl, and GSK3β and β-catenin.2+- and PCP-pathways [Regarding the Frizzled receptor family, Fdz6 is a seven transmembrane-spanning receptor whose Wnt binding regulates activation of canonical and non-canonical pathways, specifically Capathways . Fdz6 knpathways ,97. Thespathways ,97. The Dvl and β-catenin play a critical role in dendritic arborisation  and in aGenetic studies have thoroughly investigated the role of GSK3β polymorphisms and genes involved in Wnt signalling in the cerebral cortex. These studies shed light on the association between polymorphisms and various regional changes in brain structure observed in MDD patients ,101. SevRegional grey matter (GM) volume changes in MDD patients were found in temporolateral and medial prefrontal cortices, in association with SNPs in canonical Wnt signalling pathway genes and in GSK3β substrate proteins genes . GM voluOther four SNPs in the non-coding region of the GSK3β, –50 T/C, +9227 A/G, +11660 G/T and +4251 T/A have been significantly associated with 4-week antidepressant response .Post-mortem studies showed increased activity of GSK3β in suicide victims with MDD  and abnoCombining these data we may state that Fzd receptors play a role in neurogenesis, synaptic and neuronal plasticity. Thus, it is possible that they are involved in the pathogenesis of MDD. Genetic studies heretofore identified polymorphisms of genes encoding interacting proteins in brain regions involved in MDD, such as hippocampus and temporal gyrus. Genetic variations along the Wnt pathway may be not only involved in disease vulnerability, but also in treatment response. These data need replication.7.3Suicidal ideation occurs more frequently in mood disorders and death by self-killing is more frequently associated with depression, a state common to BP and MDD and the major source of burden in both disorders.The Wnt signalling pathway in suicide has not been widely studied so far. Recent post-mortem studies showed increased GSK3β and decreased Akt activities in brains of suicide victims with MDD, but not in suicide victims without depression .P<0.001; however, when these SNPs were subsequently tested for association in another large and independent bipolar sample, no one was found to be significantly associated after correcting for multiple testing. Despite this, the combined analysis of all sample sets produced an association signal at the threshold of genome-wide significance only for 2p25 (rs300774), with the associated SNPs on 2p25 falling in a large linkage disequilibrium block containing the ACP1 (acid phosphatase 1) gene, a gene whose expression was significantly elevated in people with bipolar disorder who committed suicide. The ACP1 protein is a Wnt signalling influencing tyrosine phosphatase.One genetic study tackled the issue of genetic polymorphisms and suicidality, focusing on polymorphisms of two common GSK3β SNPs, –1727A/T and –50C/T . Finding88.18.1.1Lithium is the gold standard in the treatment of BP. It is widely used in the treatment of acute manic episodes and in the prophylaxis of manic and depressive episodes. In addition, lithium possesses an independent antisuicidal action . Lithiumet al., [2+ binding site [Klein and Melton  and Hedget al.,  showed fing site . The inding site , cultureing site , and mouing site . How liting site -120. Thuing site .et al., [A series of genetic studies in an Italian population indicated that a SNP in the promoter region of GSK3β (–50T/C) was associated with BP, with the C-variant being associated with later onset and better response to lithium or acute sleep deprivation -84. In aet al.,  did not Clock [Clock-mutant mice were advanced as a genetic mouse model for bipolar mania [A role for GSK3β inhibition  in the therapeutic action of lithium in mood disorders is supported by preclinical studies. Acute administration of lithium or valproate increases β-catenin levels in the rat frontal cortex . LithiumClock . Clock-mar mania , and polar mania -129 , is an antiepileptic drug with demonstrated efficacy in the treatment of BP-associated acute mania and in the prevention of manic episodes of BP. Valproate is sometimes used to reduce the risk for manic switch in patients with bipolar depression, when antidepressants are concurrently used , despitetau protein phosphorylation-mediated GSK3 inhibition in human neuroblastoma cell lines overexpressing GSK3β [in vivo [via two different mechanisms, which results into a synergic enzyme inhibition when the two drugs are combined [in vivo studies are not homogeneous. Kozlovsky et al., [9 phosphorylated (inhibited)-GSK3β in the frontal cortex [Whether valoproate acts as an inhibitor of GSK3β is matter of debate ,138-143.ng GSK3β , or thro[in vivo . Valproa[in vivo ; howevercombined . Valproacombined ,146,147.combined . In cultcombined . Data of et al.,  showed tl cortex . In anotl cortex .et al., [The influence of valproate on the Wnt pathway is not restricted to the inhibition of GSK3β. Zhou et al.,  have fouHuman studies on valproate and the Wnt/GSK3β/β-catenin pathway have generated contrasting data. An eight-week treatment of BP patients with valproate, lithium, and/or atypical antipsychotics enhanced serine phosphorylation of GSK3 in peripheral blood mononuclear cells. Interestingly, concomitant electroconvulsive therapy prevented this effect . Post-moThus, whether and how precisely valproate influences Wnt signalling, and whether this contributes to the therapeutic efficacy of valproate in BPs, remains unclear.1).Few studies have investigated the effect of treatment with antiepileptic drugs used as mood stabilisers, such as carbamazepine or lamotrigine, on GSK3β and other Wnt-related proteins. There is only an unpublished observation that chronic lamotrigine treatment increased serine phosphorylation of GSK3 in the mouse hippocampus or cerebral cortex (reported in ). Other 8.22A serotonin receptors, and, with the exception of risperidone and 9-hydroxy-risperidone, show a low D2 dopamine receptor occupancy and are therefore devoid of extrapyramidal side effects. It is generally believed that psychosis is associated with an increased activity of the meso-limbic dopaminergic system, and a reduced activity of the meso-cortical dopaminergic system [et al., [2 receptor blockade. Others confirmed that dopaminergic drugs increase the activity of GSK3β [Antipsychotics are used in patients with schizophrenia, in the manic phase of BP, in other psychotic disorders, like delusional disorder or shared psychotic disorder, and in depression with psychotic features. Atypical antipsychotic drugs antagonize 5-HTc system -159. Bea[et al.,  showed tof GSK3β ,160, whiof GSK3β ,161-165.et al., [9 phosphorylation of GSK3 in the mouse cerebral cortex, hippocampus, striatum, and cerebellum. There was a synergism between risperidone and antidepressants (monoamine oxidase inhibitors or fluoxetine) in enhancing GSK3 phosphorylation, an evidence that supports the use of risperidone in mood disorders [9-phosphorylated GSK3, and β-catenin in the frontal cortex [9 by multiple signalling pathways including the canonical Wnt pathway and the PI3K/Akt pathway. Roh et al., [9 and Ser21; however, only clozapine increased Dvl phosphorylation. In neuroblastoma cell lines showed that clozapine increased Ser9 phosphorylation of GSK3β via the activation of the canonical Wnt pathway, but not via the activation of the PI3K/Akt pathway [via the PI3K/Akt pathway is matter of debate [inhibits Wnt signalling. This unexpected effect is secondary to increases in intracellular free Ca2+ which results from a thapsigargin-like activity of spiperone at intracellular Ca2+ stores [Clozapine, haloperidol, and risperidone were found to increase GSK3 phosphorylation, as well as Dvl-3 and β-catenin levels in the rat medial prefrontal cortex and striatum . Levels et al.,  found thisorders . Chronicl cortex . As outl et al.,  found th pathway . Whetherf debate -171. On + stores . This ad+ stores .et al., [The modulation of Wnt signalling by antipsychotic drugs might have a strong impact on the alterations of neuronal connectivity and cell fate associated with psychotic disorders. In a recent elegant study, Brennand et al.,  demonstr1). This may underlie the antimanic and mood-stabilising activity of antipsychotics in BP.Taken together, these data suggest that antipsychotics share the ability of lithium and valproate to activate Wnt signalling through the inhibition of GSK3β or other mechanisms . The relative contribution of the Wnt pathway and other signalling pathways to the dysregulation of GSK3β associated with BP or MDD remains to be established.Data suggesting a possible involvement of the Wnt signalling pathway in the pathophysiology of BP and MDD are sound, but not always consistent. We should underline that Wnt signalling has not been studied in other mood disorders, such as dysthymic disorder or mood disorder, not otherwise specified. The Wnt-related protein that has been consistently associated with BP or MDD is GSK3β; however, this molecule is at the crossroads between other signalling pathways that also appear to be involved in mood and psychotic disorders  might improve mood disorders without the adverse effects associated with the use of the current antidepressants or mood stabilisers. However, the complexity of the mechanisms underlying the pathophysiology of mood disorders might favour the use of drugs with multiple mechanisms of action over the use of small molecule inhibitors of individual enzymes. This particular issue is critical to the design of new drugs in psychiatric disorders and warrants further investigation.


Herein, we test the hypothesis that β-catenin and not necessarily NF-κB regulates colonic crypt hyperplasia or tumorigenesis in response to CR infection. When C57Bl/6 wild type (WT) mice were infected with CR, sequential increases in proliferation at days 9 and 12 plateaued off at day 19 and paralleled increases in NF-κB signaling. In −/−Tlr4 (KO) mice, a sequential but sustained proliferation which tapered off only marginally at day 19, was associated with TLR4-dependent and independent increases in NF-κB signaling. Similarly, increases in either activated or total β-catenin in the colonic crypts of WT mice as early as day 3 post-infection coincided with cyclinD1 and c-myc expression and associated crypt hyperplasia. In KO mice, a delayed kinetics associated predominantly with increases in non-phosphorylated (active) β-catenin coincided with increases in cyclinD1, c-myc and crypt hyperplasia. Interestingly, PKCζ-catalyzed Ser-9 phosphorylation and inactivation of GSK-3β and not loss of wild type APC protein accounted for β-catenin accumulation and nuclear translocation in either strain. In vitro studies with Wnt2b and Wnt5a further validated the interplay between the Wnt/β-catenin and NF-κB pathways, respectively. When WT or KO mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si- β-Cat), almost complete loss of nuclear β-catenin coincided with concomitant decreases in CD44 and crypt hyperplasia without defects in NF-κB signaling. si-β-Cat treatment to ApcMin/+ mice attenuated CR-induced increases in β-catenin and CD44 that halted the growth of mutated crypts without affecting NF-κB signaling. The predominant β-catenin-induced crypt proliferation was further validated in a Castaneus strain (B6.CAST.11M) that exhibited significant crypt hyperplasia despite an attenuated NF-κB signaling. Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia in response to bacterial infection.Both β-catenin and NF-κB have been implicated in our laboratory as candidate factors in driving proliferation in an  This hypothesis was tested in the current study.Citrobacter rodentium (CR) elicited a predictable response in the distal colon: gross thickening accompanied by hyperplasia and significant increase in crypt length between days 7 to 12 post-infection. The crypt length however, plateaued between days 12 to 19  and −536 (p65536) phosphorylation of p65 subunit, relative levels of both crypt cellular and nuclear p65276 and p65536 along with total p65 increased significantly between days 3–12 compared with uninfected control while relative levels of both moieties declined at day 19  increased between days 5–12 post-infection compared to uninfected control before declining at day 19 (i) and correlated with gross changes in hyperplasia. Increases in Ser45-phosphorylation when normalized to total β-catenin levels however exhibited only subtle enhancement in phosphorylation intensity suggesting that increases in β-Cat45 may be due to a proportional increase in overall β-catenin abundance (i). Similarly, relative levels of β-cat552 as well as de-phospho-β-catenin (dβ-Cat), both known to represent active β-catenin, increased significantly in both the cellular and nuclear extracts between days 5–12 compared to uninfected control before a rapid decline to basal levels at day 19 (i). As before, the increases in these species when compared to changes in total β-catenin were subtle. The alterations in various β-catenin species correlated with relative levels of downstream targets c-myc and cyclinD1 (ii). In −/−Tlr4 mice, increases in cellular and nuclear levels of β-Cat45 and β-Cat552 at days 5 and 12 compared to uninfected control (i), were mirror images of changes recorded in phosphorylated p65 at these time points  were similar to those recorded in the crypts of wild type C57Bl/6 mice (see i) except that the levels even at day 19 were significantly higher than uninfected control. The increases in these species were apparently a reflection of an overall increase in total β-catenin at these time points but unlike wild type crypts wherein, the total β-catenin returned to baseline at day 19, total β-catenin in −/−Tlr4 crypts remained elevated than uninfected control (i). When we measured downstream targets, both c-myc and cyclinD1 exhibited kinetics similar to that recorded in wild type crypts except that the levels even at day 19 were still higher than uninfected control (ii). These changes correlated well with persistence of crypt hyperplasia at this time point .Next, we examined the contribution of β-catenin towards crypt hyperplasia in response to CR infection in the wild type and −/−Tlr4 mice suggesting that increases in β-catenin at day 12 were not due to loss of wild type APC (Tlr4−/− mice compared to the wild type counterpart (−/−Tlr4 mice suggesting (but certainly not proving) PKCζ-mediated inactivation of GSK-3β at this time point while changes in wild type mice were less significant . These increases were either at par or even higher than those recorded with CR infection (i). Interestingly, we did not observe any detectable levels of either p65 phosphorylated at Ser-536 (p65536) or β-catenin’s downstream targets Slug and Snail following addition of purified Wnt2b while CR infection alone promoted increases in these proteins at indicated times (i). Wnt5a addition on the other hand, led to significant increases in relative levels of nuclear p65536 at 24 hr which persisted until 48 hr before declining at 72 hr similar to those recorded with CR infection alone while Wnt5a only had nominal effect on β-catenin levels at these time points (ii). Similar to Wnt2b, Wnt5a did not affect Slug and Snail expression at either time point (ii). Next, during measurement of NF-κB activity, CR infection compared to uninfected control, caused a reproducible increase in NF-κB activity at 24 hr which persisted until 72 hr  of purified Wnt2b and Wnt5a proteins in modulating the Wnt and NF-κB pathways ed cells Fig. 5A,in vivo in response to CR infection in the wild type (WT) mice and mice deficient for TLR4. In response to either CR infection or CR infection+vehicle treatment of either strain, significant increases in both active and total β-catenin was observed in the crypt cellular and nuclear extracts along with increases in its downstream target cyclinD1, compared to uninfected control (Tlr4−/− mice were treated with nanoparticle-encapsulated siRNA to β-catenin (si-β-Cat), we observed almost complete loss of nuclear β-catenin with concomitant decreases in cyclinD1  or total NF-κB-p65 which remained elevated in CR infected crypt cellular and nuclear extracts . Thus, β-catenin and not necessarily NF-κB regulates crypt hyperplasia and tumorigenesis in response to CR infection.Our next aim was to understand the interplay between the β-catenin and NF-κB pathways Bacillus anthracisi). In CAST.11M mice however, Ki-67 staining and crypt lengths at day 12 and particularly at day 19 compared to uninfected controls were 1.5–2 fold higher than those recorded in C57Bl/6 mice at the same time points (ii). Transmission electron microscopy revealed no difference in bacterial binding at the luminal surface in either strain  and recruitment of CD3+ T-cells and F4/80+ macrophages to the sub-epithelial regions compared to CAST.11M mice . Mechanistically, in C57Bl/6 mice, Ki-67 kinetics coincided with increases in nuclear accumulation of β-catenin and its downstream target cyclinD1 at days 9 and 12 compared to uninfected control while the levels declined at day 19 . In addition, p65276/p65536 subunit expression (i) and NF-κB activity (ii) increased significantly at days 9 and 12 with declining trend at day 19 that correlated with downstream target CXCL-1 expression (i). In CAST.11M mice on the other hand, we observed a delayed response wherein, nuclear β-catenin levels did not go up until day 12 with sustained expression at day 19 (i) which coincided with cyclinD1 expression (ii) and Ki-67 kinetics at these time points. Interestingly, CAST.11M mice did not exhibit any significant increase in phosphorylated/total p65 subunit expression (i), NF-κB activity (ii), CXCL-1 expression (ii) or serum FITC-Dextran levels (Suppl.Castaneus mice contain a CAST/Ei region on chromosome 11 (CAST.11M) on an otherwise C57BL/6 genetic background. Genetic variation within an 8.3 Mb region on mouse chromosome 11 controls host response to anthrax lethal toxin and resistance to infection by the Sterne strain of Wnt/β-catenin and NF-κB are independent pathways involved in the regulation of physiological and pathological effects related to the development, immune function, inflammation, tumorigenesis and frank malignancy. The recent discovery of functional cross-regulation between the two pathways has established complex roles for Wnt/β-catenin and NF-κB signaling in the pathogenesis of various diseases including cancer. Indeed, the mechanisms and biological effects of the cross-regulation of the two pathways remains an area of intense investigation. It is now evident that strategies targeting the cross-regulation between these pathways may be a promising direction for future cancer therapeutics.Tlr4−/− mice in the B6 background wherein, initial kinetics of NF-κB activation at day 3 highlighted the need for the presence of TLR4; NF-κB activation at day 5 however, may represent a homeostatic response to counter bacterial infection while NF-κB activation at day 12 which correlated with peak hyperplasia  by toll like receptor-4 (TLR4) and subsequent activation of NF-κB not only activates host defense against pathogenic bacteria, but can also provide mucosal protection. In pathological situations however, TLR4 is upregulated in both Crohn’s disease and ulcerative colitis, two components of human IBD, leading to pro-inflammatory signaling via NF-κB in vitro. The same group previously showed that TLR4 signaling in the colon induces epithelial proliferation and protection against apoptosis Tlr4−/− mice and these changes directly correlated with increases in crypt hyperplasia in infected mice. A definitive role for β-catenin in CR-induced crypt hyperplasia in Tlr4−/− mice was established by blocking increases in β-catenin in vivo which resulted in attenuation of the hyperplastic response. These results clearly contradict the notion that TLR4 is somehow required for activation of the Wnt/β-catenin pathway. Infact, a recent study by Sodhi CP et al. Tlr4-deficient mice corroborate with those described during NEC, the regulation of Wnt/β-catenin signaling via TLR4 seems more complex than what was initially envisioned.In addition to TLR4’s contribution towards NF-κB activation in response to Gram negative bacteria or LPS, TLR4 has also been involved in modulating the Wnt/β-catenin pathway in the intestine, and that over-expression of TLR4 has been found sufficient for priming the intestinal mucosa towards neoplasia. Indeed, Santaolalla R. et al. −/−Tlr4 mice or in ApcMin/+ mice, knockdown of β-catenin significantly impacted both hyperplasia and tumorigenesis in response to CR infection. Since a combination of canonical and non-canonical Wnt signaling regulates the interplay between the Wnt/β-catenin and NF-κB pathways in vitro. Based on functional assays with purified proteins, we conclude that Wnt2b but not Wnt5a regulates the Wnt/β-catenin pathway while Wnt5a and not Wnt2b is involved in the activation of the NF-κB pathway, respectively. In previous studies, Wnt2b is shown to be transiently expressed in the primitive streak during gastrulation and has been suggested to primarily play a supportive role in gastrulation and organogenesis in vitro, studies in new mouse models allowing specific deletion of genes encoding these proteins in the epithelial cells of the colon will be required to address the potential role(s) of these ligands in either crypt hyperplasia or tumorigenesis in response to CR infection.Continuing with our theme of interplay between the Wnt/β-catenin and the NF-κB pathways, an elegant study recently reported that constitutive IKKβ/NF-κB activation strongly synergized with Wnt signaling to promote intestinal tumorigenesis The dominance of β-catenin-induced crypt proliferation in response to CR infection was further validated in a Castaneus strain (CAST.11M) that exhibits high susceptibility to Anthrax’s lethal toxin (LT) Helicobacter pylori-free ApcMin/+ and −/−Tlr4 mice were procured from Jackson Laboratory, Bar Harbor, Maine, USA. B6.CAST.11M Castaneus mice were kindly provided by Drs. Aldons J. Lusis and Richard C. Davis  and the generation of these mice has been described previously ApcMin/+, −/−Tlr4 and wild type C57Bl/6J mice by oral inoculation with a 16-h culture of CR  identified as pink colonies on MacConkey agar, as previously described This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All protocols were approved by University of Kansas Medical Center Animal Care and Use Committee. All efforts were made to minimize suffering. Male 5′-CACCCGGACTGATCTTGTCT-3′; R: 5′-TGTTTCTGCACTCCTTGCAC-3′) and Wnt5a  were identified by performing semi-quantitative PCR using 1/20 dilution of the cDNA. For each gene product, the amplification cycle number was chosen empirically within the linear range. The PCR products were separated by polyacrylamide gel electrophoresis and visualized by ethidium bromide staining of the gels under UV light. Gel data were recorded with the Bio-Rad FluorS Imaging System, and relative densities of the bands were determined with Quantity One software . Gene expression was normalized with β-actin expression. Total RNA samples were also subjected to real-time polymerase chain reaction (PCR) by SYBR chemistry  using primers specific for CXCL-1/KC  and GAPDH . The changes in mRNA were expressed as fold change relative to control with ± SEM value.Total RNA was extracted from isolated colonic crypts by using TRI Reagent. Following cDNA synthesis using Superscript II and random primers, gene products specific to Wnt-2b (F: 2 at 33°C. YAMC cells (5×105) were transfected with either 100 nmol/L of scrambled siRNA or siRNAs specific for Wnt2b (sc-155356) and Wnt5a (sc-41113) using 2–8 µl of transfection reagent  for 24 h. CR strain DBS 100 (ATCC cat.# 51459™) was grown under aerophilic conditions on Luria-Bertani (LB) agar plates for 24 h at 37°C and cultured in LB broth O/N at 37°C. RPMI, containing 0.45% glucose was inoculated with a 1∶20 dilution of a standard LB overnight culture and incubated for 2 h at 37°C. Monolayers of 5 105 YAMC cells at ∼50% confluence or cells transfected with various siRNAs after 24 h were infected with CR at a multiplicity of infection (MOI) of 90 or the medium alone (as a control) or incubated with purified Wnt2b and Wnt5a (@250 ng/ml) for 3 h at 37°C in 5% CO2. After 3 h, medium was changed and replaced with fresh medium plus antibiotics to ensure complete absence of live bacteria. Relative levels of activated p65-NF-κB in the nuclear extracts were measured using the TransAm p65 NF-κB Chemi Transcription Factor Assay Kit from Active Motif  as per manufacturer’s instructions.For DNA binding assay, nuclear extracts were prepared from the distal colonic crypts of uninfected, or days-3, 5, 7, 9, 12 and 19 infected mice, respectively. The young adult mouse colon (YAMC) cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 µg/ml gentamicin, 100 units/ml penicillin, 100 µg/ml streptomycin, and 5 units/ml IFNγ in a humidified incubator with 5% CO@1∶90 MOI) or incubated with purified Wnt2b and Wnt5a (@250 ng/ml) at 37°C in 5% CO2 for 3 hr followed by washing to remove bacteria. At indicated times, cells were processed for measurement of luciferase activity with a luminometer and the values were normalized to the internal control. All transfection experiments were repeated at least three times.Transfection experiments were carried out using Lipofectamine 2000 reagent (Invitrogen Life Technologies), according to the manufacturer’s instructions. YAMC cells were maintained as described above. For transient transfections, cells were: i) co-transfected with Tcf-4 reporter plasmid  or a mutant Tcf-binding site  along with pRL-TK Renilla vector as internal control, or ii) transfected with either 100 nmol/L of scrambled siRNA or siRNAs specific for Wnt2b (sc-155356) and Wnt5a (sc-41113) using 2–8 ml of transfection reagent . Twenty four hour post-transfection, cells were either uninfected (medium only) or infected with CR  acid nanoparticles (siβ-Cat-NPs) (at 4 µM/Kg. body wt.) every alternate day for 10-days followed by euthanasia at 12-days post-infection For blocking β-catenin 276, β-catenin, cyclinD1, CD44, CD133 and Dclk1 were performed on 5-µm-thick paraffin embedded sections prepared from the distal colons of uninfected normal, 3, 5, 7, 9, 12 and 19 days CR-infected or CR-infected and siβ-Cat-NPs treated mice utilizing the HRP labeled polymer conjugated to secondary antibody using Envision+System-HRP  with microwave accentuation as described previously t-test was performed as described below.For histology, tissues were fixed with 10% neutral buffered formalin or in Carnoy’s fixative  prior to paraffin embedding. Paraffin-embedded sections (5 µm) were stained with H&E for gross morphology and the pictures were obtained with an Eclipse E1000 microscope (Nikon). IMF to detect β-catenin in YAMC cells treated as described above or IHCs to detect Ki-67, p65@90∶1 MOI) for 3 h followed by washing to remove bacteria. At 48 h, cells were wounded with a plastic pipette tip. After removing debris and adding fresh media, cell migration was followed for 12–16 h YAMC cells were transfected with siRNA specific to Wnt2b and Wnt5a, respectively for 24 h. Cells were subsequently treated with control media or with CR  for multiple group comparisons . p-values <0.05 were considered statistically significant.Experiments were repeated three times with consistent results. Data were expressed as mean values ± standard error. Statistical analyses for all studies were performed using unpaired, two-tailed Student’s Figure S1Effect of CR infection on NF-κB-p65 phosphorylation in −/−Tlr4 mice. Representative photomicrographs of paraffin embedded sections prepared from the distal colons of uninfected normal (N) and days 9–19 post-CR infected −/−Tlr4 mice and stained with H&E for gross morphology (left panel) and for p65 subunit phosphorylated at Ser-276 (p65276). Percentages represent percent cells positive for p65276. Scale bar: 100 µm; n = 3 independent experiments.(TIF)Click here for additional data file.Figure S2A. Effect of MEK inhibition on cell migration. A representative bar graph showing percent migration at 16 h . B. Effect of CR infection on paracellular permeability. FITC-Dextran Assay. Uninfected normal (N) and CR infected C57Bl/6 (C57) or B6.CAST.11M (11M) mice were subjected to gavage with FITC-D, and serum concentrations, shown as fluorescence units, were measured 4 h later .(TIF)Click here for additional data file.Figure S3Effect of CR infection on recruitment of inflammatory cells. Representative photomicrographs of paraffin embedded sections prepared from either the distal colons or crypt-denuded lamina propria (CLP) of uninfected normal (N) and days 9–19 post-CR infected C57Bl/6 or B6/CAST.11M mice and stained with antibodies for: CD3+ T cells (upper panel) or F4/80+ macrophages (lower panel). Scale bar: 50 µm; n = 3 independent experiments.(TIF)Click here for additional data file.


Moreover, the introduction of Nef into HEK293 cells specifically inhibited a Wnt pathway reporter. The Wnt signaling pathway is implicated in major physiologic cellular functions, such as proliferation, migration, cell fate specification, maintenance of pluripotency and induction of tumorigenicity. Proliferation and migration are important responses of T-cells, which are major cellular targets of HIV infection. Using an informatics screen, we identified a previously unsuspected interaction between HIV’s Nef protein and β-catenin, a key component of the Wnt pathway. A segment in Nef contains identical amino acids at key positions and structurally mimics the β-catenin binding sites on endogenous β-catenin ligands. The interaction between Nef and β-catenin was confirmed  This motif captures the precise three-dimensional structural profile specific for the central region of β-catenin binding. Notably, the first position of the pattern is restricted to aspartate as this side chain is snugly buried in a highly specific β-catenin surface pocket and no substitutions are seen in any of the bound ligands. The same is true for the sixth position where the phenylalanine or tyrosine occupy a slot-like pocket restrictive for a benzene aromatic ring. The motif was then used as an input for a MyHits “pattern search” (http://myhits.isb-sib.ch/cgi-bin/pattern_search)  [β-catenin co-crystal structures  –35 were : 10239] .http://www.hiv.lanl.gov/content/sequence/NEWALIGN/align.html). The percentage of Nef sequences carrying the D-x-x-x-x-[FY] motif were calculated with a custom Perl script (available upon request from the authors).Nef sequences from SIV, HIV2 and HIV1 subtypes were downloaded from the HIV Sequence Alignment tool incorporated in the HIV LANL database, with only one sequence per patient selected  were used for the docking simulation. Hydrogen atoms were added to the crystal structure and the structure was then converted into an internal coordinate representation according to the ICM method . Full-atEscherichia coli BL21 (DE3) harboring pGEX-4T-2-NEF   were grown in LB medium containing 100 μg/ml ampicillin at 18 °C. At the exponential phase of growth (OD600=0.6), isopropyl- β -Dthiogalactopyranoside (IPTG) was added to the culture medium at a final concentration of 0.8 mM. After an additional 12 hours of culture, cells were harvested by centrifugation (4200 rpm for 15 minutes). The bacterial pellet was suspended in lysis buffer , sonicated on ice and centrifuged at 4 °C, 13,200 rpm for an additional hour. The supernatant was incubated for 1.5 hours with glutathione beads. The beads were washed three times with wash buffer  and an additional fourth wash . The beads were eluted with 50mM glutathione buffer .Escherichia coli BL21 (DE3) harboring pExHTB-β-catenin  were grown and induced as described above. The cell paste was then suspended in lysis buffer , followed by sonication on ice and centrifuged at 4 °C, 13,200 rpm for an additional hour. The supernatant was incubated for 1.5 hours with Ni beads. The beads were washed three times with wash buffer  and an additional fourth wash . For the purpose of the pull down assay, β-catenin was not eluted off the Ni beads.Purified GST-Nef was added to β-catenin that was immobilized on Ni beads as described above. The proteins were incubated together for 2 hours and washed three times with wash buffer  and a fourth wash . Beads were eluted with 250mM NaCl, 25mM TrisHcl pH8, 1% glycerol, 1mM PMSF, 1% triton, 15mM β-mercaptoethanol, 600mM imidazole) for 1h. Protein complexes bound to the beads were eluted and denatured by the addition of SDS. The samples were then loaded on a 10% SDS denaturing gel and transferred overnight into a nitrocellulose membrane. Proteins were detected by a western blot. β-catenin was detected by using a monoclonal mouse antibody . GST-Nef was detected by using a rabbit polyclonal rabbit anti GST antibody . Goat peroxidase-conjugated anti-rabbit or anti-mouse antibodies  were used and binding was detected by enhanced chemiluminescence . The experiment was repeated three times and a representative blot is shown in 2.HEK293 cells were cultured in Dulbecco’s Minimal Essential Medium (Invitrogen), supplemented with 10% fetal bovine serum and sodium pyruvate. Cells were incubated in a 37°C incubator in an atmosphere of 5% COExpression vectors encoding WT-Nef, D186A-Nef and F191A-Nef were transfected into 293 cells in 10cm plate with Effectene (Qiagen). The expression vector was obtained through the NIH AIDS Research and Reference Reagent program from Drs. Yingying Li, Feng Gao, and Beatrice H. Hahn (Cat# 6173). Cells were incubated for 16-18 hours post-transfection, washed 4 times with cold phosphate buffered saline (PBS), lysed with 1ml lysis buffer , 1mM Na3VO4, 5mM EDTA, 1mM NaF, protease inhibitor) and put on ice for 2 hours. Cell suspensions were centrifuged for 10 minutes at 13,200 rpm, and the supernatants were incubated with monoclonal anti-β-catenin antibody  or with normal mouse IgG (Santa-Cruz). After 16-20 hours, 25 μl of protein A/G beads (Santa-Cruz) were added to the antibody-cell suspension solution for an additional 2 hours. Beads were washed with wash buffer  four times by inverting the tubes three times followed by 5 minute incubation on ice and another three washes. 100μl of 3X sample buffer was added to beads and incubated on ice for 1.5 hours. Beads were boiled for 5 minutes and supernatants were resolved on a 12% acrylamide gel by electrophoresis at 90V. Gels were presoaked in transfer buffer  for 30 minutes at 4 degrees C and then transferred to nitrocellulose membranes  using a transfer cell (Bio-Rad). Nitrocellulose membranes were blocked with 5% fat-free dried milk in TBST  for one hour at room temperature (RT). Membranes were incubated with primary antibody  diluted in 2% milk in TBST (1:1000 dilution) for one hour at RT, followed by three washes with TBST for 10 minutes each. Secondary HRP antibodies (R&D) were then added (1:1000 dilution) one hour at RT with gentle agitation on a rocking plate. The signal was detected using ECL reagents (Pierce).ONE-HOUR IP-Western Kit (Genscript) that specifically blocks light and heavy chain contamination in IP experiments was used to detect Nef. Nef antibody #1539  was use293 cells were transfected with 15ng TopFlash or FopFlash, 5ng CMV-Renilla and with either empty vector or 50ng of codon-optimized WT-Nef, or Nef mutants. The transfection mix was first added to each well in the 96-well plate and only then 293 cells were added to the plate. The FopFlash plasmid contains TCF binding sites which are mutated in the FopFlash plasmid. Transcription from this reporter plasmid is activated when β-catenin enters the nucleus and binds TCF. The TCF/β-catenin complex then binds to the TCF binding sites on the reporter to initiate transcription of Firefly luciferase. Cells were treated with Wnt-conditioned media to activate the Wnt pathway. 5 hours post transfection, cells were replenished with fresh Wnt-conditioned media for an additional 10 hours. The assay was performed by using the Promega Dual-Luciferase® Reporter Assay System. It is important to note that that WT-Nef and the two mutants affect Renilla’s trancription levels to the same degree .Data File S1http://myhits.isb-sib.ch/cgi-bin/pattern_search).The file documents the output of the “pattern search” tool implemented in the MyHits website Click here for additional data file.Data File S2Row data for luciferase assay in which 293 cells were transfected with 15ng TopFlash or FopFlash, 5ng CMV-Renilla and with either empty vector or 50ng of codon-optimized WT-Nef, or Nef mutants.(XLS)Click here for additional data file.Figure S1Coomasie Blue staining of three different pull down control experiments.A. Pull down experiments of His-β-catenin and WT-Nef. “w” stands for washing step and “EL” stands for elution B. pull down experiment of His-β-catenin and GST C. Incubation of WT-Nef-GST and Ni beads.(TIF)Click here for additional data file.Figure S2Cytoplasmic and nuclear fractionation of HEK293 cells transfected with different amounts of WT-Nef.HEK293 cells were plated in a 10cm dish and transfected 24h later with an empty vector or WT-Nef encoding plasmid up to 2ng. The cells were lysed 15 hours post transfection. The nucleus (lanes 1-5) and cytoplasm (lanes 6-10) were extracted using the NE-PER kit by Thermo-Scientific. The cytoplasmic and nuclear fractions were blotted with anti-tubulin, a cytoplasmic marker and anti- Histone H1, a nuclear marker, in order to assess the purity of the cytoplasmic and nuclear lysates. The lysates were also blotted with anti-actin antibody as a loading control. Uppermost panel: immunoblot of endogenous β-catenin using mouse anti- β-catenin Ab for detection. 2nd to top panel: immunoblot of different amounts of transfected WT-Nef (0-2ng) using mouse anti-Nef Ab for detection. 3rd to top panel: immunoblot of alpha-tublin using anti-alpha tubulin Ab for detection. Lowest panel: immunoblot of actin using anti-pan-actin for detection.(TIF)Click here for additional data file.Table S1Contact areas of Nef residues in the β-catenin binding motif upon docking to β-catenin.(DOC)Click here for additional data file.


The Wnt signaling pathway is involved in a wide range of embryonic patterning events and maintenance of homeostasis in adult tissues. The pathological role of the Wnt pathway has emerged from studies showing a high frequency of specific human cancers associated with mutations that constitutively activate the transcriptional response of these pathways. Constitutive activation of the Wnt signaling pathway is a common feature of solid tumors and contributes to tumor development, progression and metastasis in various cancers. In this review, the Wnt pathway will be covered from the perspective of urological cancers with emphasis placed on the recent published literature. Regulation of the Wnt signaling pathway by microRNAs (miRNA), small RNA sequences that modify gene expression profiles will also be discussed. An improved understanding of the basic genetics and biology of Wnt signaling pathway will provide insights into the development of novel chemopreventive and therapeutic strategies for urological cancers. Hydra to Xenopus [Drosophila and animal models and have been implicated in human cancers. Other signaling pathways important in embryonic pattern formation include the Nothch pathway and the tyrosine kinase receptor/Ras pathways [During embryogenesis, cells often acquire new identities as they migrate to new locations. Many of these morphogenetic changes are induced by extracellular ligands and their receptors -3. Signa Xenopus ,15. Defepathways  and thospathways ,18. Instpathways -21. Althpathways ,22-25.Drosophila segment polarity gene Wingless (Wg) and the murine protooncogene Int-1 [http://www.stanford.edu/~rnusse/wntwindow.html). Intensive studies from past decades have identified essential components of signaling pathways by which Wnt proteins relay their signals into intracellular responses [Wnt-1, the first member of Wnt family protein was initially identified independently as a ne Int-1 . The terne Int-1 . For a wesponses ,27. Wnt esponses -30.Wnt-1 as Int-1, a gene activated by nearby integration of the mouse mammary tumor virus in a mammary tumor model [APC or stabilizing oncogenic mutations in β-catenin [Early evidence of involvement of the Wnt pathway in cancer came from isolation of or model . Oncogenor model ,33. Thesor model . Many muor model . Moreoveor model ,37. Muta-catenin ,38. Loss-catenin . These e-catenin . These p-catenin . To furt2-terminus of cytosolic β-catenin is constitutively phosphorylated by a dual kinase mechanism. CK1-α phosphorylates β-catenin at Ser45, and this priming phosphorylation results in subsequent phosphorylaion by GSK3-B at residues Thr41, Ser37, and Ser33 [Myc oncogene [Myc also shows copy number amplification in a subset of primary ccRCC [Renal cell carcinoma (RCC) is the most common type of kidney cancer, accounting for 90% of all kidney cancers. It can be further classified into clear cell , papillary 10-15%), chromophobe (5%), collecting duct (very rare) and a remaining unclassified group (5%) . The ultnd Ser33 ,44. β-cand Ser33 . Wnt posoncogene   in aggressive RCC [The genetic basis of RCC is associated with the von Hippel-Lindau (VHL) tumor suppressor gene, identified in 1993 . This gesive RCC . Wnt hassive RCC . The tum2+ and Wnt/polarity, also known as Planar Cell Polarity (PCP) pathways are known as the "non-canonical pathways". Other non-canonical pathways include Wnt/Jnk and Wnt/Rho signaling [2+ pathway regulates cell adhesion and motility, and is mediated through release of intracellular Ca2+ upon Wnt stimulation [2+ pathway activated by Wnt-5a, antagonizes the Wnt/β-catenin pathway [The Wnt/Caignaling ,30. The ignaling . The Wntmulation ,75. Wnt-mulation . Interes pathway -80. In t pathway ,30,74. F pathway . Aberran pathway .The Wnt signaling pathway antagonists have been studied extensively in development and their involvement in oncogenesis has been demonstrated. There are four families of Wnt antagonists that can be divided into two sub-groups according to their mode of action. The first group includes the secreted-Frizzled related protein (sFRP) family, Wnt-inhibitory factor (WIF-1), and Cerberus. They inhibit Wnt signaling by direct binding to Wnt molecules. The second group, consisting of the Dickkopf (DKK) family, inhibits Wnt signaling by binding to the LRP5/LRP6 component of the Wnt receptor complex -85. In mAPC, AXIN1/2 and β-catenin are well established in colorectal [APC, AXIN or β-catenin are substantially less frequent. In renal cell carcinoma APC and β-catenin mutations are uncommon events [in-vitro and in-vivo [MMP10 [MMP-3 and ANGPT1 genes in renal cancer and thus contributed to the invasive capability of RCC [Abnormal promoter methylation of tumor suppressor genes contributes to tightly heritable gene silencing and can cause loss of gene function, which thereby contributes to tumorigenesis. Various Wnt pathway components that regulate proper WNT/β-catenin signaling are frequently disrupted in human cancers through either genetic or epigenetic alterations ,93. Conslorectal ,38,96,97lorectal , hepatoclorectal  as well lorectal . Most ofn events ,101. When events ,102-105.n events  highlighn events  has beenn events . Loss ofn events  than othn events . The expn events . sFRP-5 n events . The Wntn events . The Dic in-vivo ,23,109,  in-vivo . The bip in-vivo . The auto [MMP10 . sFRP-2 o [MMP10  whereas y of RCC . Uren ety of RCC . Rubin ey of RCC . The funThere is mounting evidence that aberrant activation of the Wnt pathway is frequently associated with tumor development, progression and metastasis in prostate cancer ,115. ThiA number of previous studies have reported that β-catenin is a biomarker in prostate cancer. Although β-catenin stability is regulated by a multi-component destruction complex, mutational alterations of β-catenin or other components of the destruction complexes are rare in prostate tumors. Therefore, β-catenin may be regulated by another protein in prostate cancer. Somatic deletion analysis in prostate cancers revealed a 1.4-Mb candidate tumor suppressor locus on 8p23.1, which includes the Sox7 gene . Sox7 prin-vitro and pretreatment of prostate cancer cells with DKK1 diminished osteoblast differentiation. The authors concluded that prostate cancer promotes osteoblast differentiation through canonical and noncanonical Wnt signaling pathways that stimulate both BMP-dependent and BMP-independent osteoblast differentiation [In addition to roles in the initiation and progression of the primary tumor, the Wnt pathway may also play key roles in the metastasis of prostate tumors, particularly to the bone ,126,127.ntiation . In the ntiation  proposedntiation   expression that correlated with higher tumor stage in bladder tumors . AnotherMicroRNAs (miRNAs) are small, non-coding RNAs that have been found to regulate expression of 60% of all human genes through targeted repression of gene transcription and translation. They play important roles in a wide spectrum of biological processes, including development, proliferation, and apoptosis -143. InvDespite great advances in other cancers, miRNAs related to Wnt signaling remain largely uncharted with regard to urological cancers. Signaling complexes are highly dynamic, ephemeral and non-stoichiometric molecular ensembles and emerging evidence suggests that miRNAs translate into dose-dependent responsiveness of cells to signaling molecules such as Wnt, Notch etc. As such, signaling molecules are the ideal targets for the degree of quantitative fluctuations imposed by miRNAs. This might enable the multi-gene regulatory capacity of miRNAs to remodel the signaling landscape, facilitating or opposing the transmission of information to downstream effectors in an effective and timely manner. Much work needs to be done to decipher miRNA function with regard to Wnt signaling molecules in urological cancers.Given the critical roles of Wnt pathway activation in the pathophysiology of many human diseases including cancer, interest in the development of Wnt signaling inhibitors has increased substantially. Several groups have identified various therapies and phytochemicals that either directly or indirectly disrupt β-catenin-mediated Wnt signaling. These agents include non-steroidal anti-inflammatory drugs, exisulind, vitamin A derivatives, endostatin etc. and phytochemicals such as flavanoids (genistein), curcumin, epigallocatechin-3-gallate (EGCG), resveratrol, lupeol, retinoids, lycopene and deguelin etc. . DiGiven the similarities between embryonic growth control, dysregulated cell proliferation in development of cancer and the findings that Wnt signaling regulates kidney organogenesis, the Wnts were candidates for involvement in the development of kidney cancers and thus it has been widely studied in relation to kidney cancers so far. Intense efforts have also been made recently to examine the role of the Wnt pathway in prostate cancer. The Wnt signaling pathway and its key component β-catenin have also recently emerged as important players in bladder tumorigenesis. However, very few studies have been carried out to understand the biological role of this pathway in the pathogenesis of bladder cancer. The intent of this review was to highlight the studies performed around the Wnt signaling pathway in urological cancers and to promote its unexploited potential for drug design and biomarker use.Recent data have shown that perturbations in Wnt signaling are involved in urological cancers. Tumor promotion by this pathway can proceed through a number of different genetic and epigenetic defects. In particular, a wealth of evidence implicates that chronic activation of β-catenin signaling is common in many tumor types. There is a clear need for new lead compounds targeting the Wnt/β-catenin pathway. These inhibitors may provide significant therapeutic benefit against a variety of human diseases in which the Wnt signaling pathway plays an important pathological role. Due to the emerging role of miRNAs in signal transduction, it also becomes apparent how the highly dose-sensitive nature of developmental signaling pathways like Wnt renders them prime candidates for miRNA regulation. Merging activity-based or expression-based screens with new RNA-based therapeutics may offer opportunities for targeting Wnt signaling pathways in cancer.The authors declare that they have no competing interests.SM prepared the manuscript. SS assisted in the preparation and RD edited and modified this review article. All authors read and approved the final manuscript.Dr. Shahana Majid serves as an Assistant Research Scientist in the Department of Urology at UCSF and the VA Medical Center. Dr. Majid's research interests include genetic and epigenetic studies and exploring the role of tumor suppressor genes in urological cancers. Currently, Dr. Majid's research explores the role of microRNA mediated gene activation and function in prostate, kidney and bladder cancers.Dr. Saini is an Assistant Research Scientist in the Department of Urology at SFVAMC/UCSF. Her research is primarily focused on understanding the etiology of various urological malignancies, including kidney cancers. She is particularly keen on exploring the molecular basis of progression and metastasis of these malignancies. A molecular biologist by training, Dr. Saini has worked as a post-doc fellow at University of Massachusetts Medical School Worcester and SFVAMC/UCSF.Dr. Rajvir Dahiya is a professor and Director of Urology Research Center at the UCSF/VAMC. Dr. Dahiya has a long-standing interest in molecular genetics and epigenetics of urological malignancies with a focus on developing novel biomarkers for the early diagnosis and prognosis of these cancers. Dr. Dahiya has published over 300 original research manuscripts in national and international journals and holds multiple oncology patents. Dr. Dahiya is a program director for many research projects.