There are limited therapeutic options for hepatocellular carcinoma (HCC), the most common liver malignancy worldwide. Recent studies have identified the Frizzled-7 receptor (FZD7), important for activation of Wnt-mediated signaling, as a potential therapeutic target for HCC and other cancers.We hypothesized that the extracellular domain of FZD7 (sFZD7) would be a clinically more relevant therapeutic modality than previously studied approaches to target FZD7. We expressed and purified sFZD7 from E. coli, and tested its functional activity to interact with Wnt3, its ability to inhibit Wnt3-mediated signaling, and its potential for combinatorial therapy in HCC.In vitro, sFZD7 dose-dependently decreased viability of three HCC cell lines , but had little effect on normal hepatocytes from three donors . When combined with doxorubicin, sFZD7 enhanced the growth inhibitory effects of doxorubicin against HCC cells in vitro, and against Huh7 xenografts in vivo. Reduced expressions of c-Myc, cyclin D1, and survivin were observed in vitro and in vivo. Additionally, sFZD7 altered the levels of phosphorylated AKT and ERK1/2 induced by doxorubicin treatment in vitro, suggesting that several critical pathways are involved in the chemosensitizing effect of sFZD7.sFZD7 pulled down Wnt3 from Huh7 cells, and decreased β-catenin/Tcf4 transcriptional activity in HCC cells. We propose that sFZD7 is a feasible therapeutic agent with specific activity, which can potentially be combined with other chemotherapeutic agents for the improved management of HCC. The Wnt/β-catenin signaling pathway is commonly dysregulated in various cancers, including hepatocellular carcinoma (HCC) . AberratFZDs are frequently upregulated in tumor cell lines and tissues . All tenvice versa). This interaction is indispensable for the activation of Wnt/β-catenin signaling, and interference with this interaction offers a feasible approach to modulate Wnt/β-catenin activation in cancers. SFRPs, encoding only the extracellular domain of FZD, act as natural antagonists of the Wnt/β-catenin pathway by binding to Wnt ligands and inhibiting their interactions with FZDs 2. Mice in the PBS group were sacrificed after 14 days, whereas mice in other treatment groups were sacrificed after 17 days of treatment. Tumor tissues were harvested for immunohistochemistry as described below, and for TUNEL staining using the ApopTag Peroxidase in Situ Oligo Ligation Apoptosis Detection Kit  according to the manufacturer's protocol. We assessed the  percentage of cells that stained positively with brown nuclei in five randomly selected  areas of 100 cells per area.  Western blot was used to quantify the protein levels of c-Myc, cyclin D1, or survivin in tumor xenografts of two mice in each group. The antibodies used were: HRP-conjugated anti c-Myc , biotin-conjugated anti-cyclin D1 , HRP-conjugated anti-survivin , HRP-conjugated β-actin , and secondary antibodies (anti-biotin) conjugated with HRP . Western blots were analyzed by ImageJ software, and signal intensities normalized to β-actin.Animal experiments were approved by the Administrative Panel on Laboratory Animal Care at Stanford University. Nude mice  at age 4-6 weeks (body weight of 18 to 25 g) were used. Mice were injected subcutaneously at the dorsal region with 5 × 10Immunoperoxidase staining of tumor xenografts was performed on formalin-fixed 4-μm tissue section. Briefly, sections were incubated with monoclonal mouse anti-human c-Myc , cyclin D1 , or survivin  and then washed with PBS. Subsequent procedures were performed using Dakocytomation Envision System-HRP mouse system  according to the manufacturer's protocol.Statistical significance was determined by the independent-sample T-test using the computer SPSS software . P values less than 0.05 were considered statistically significant.FZD7: Frizzled-7; HCC: Hepatocellular carcinoma; PI: propidium iodide; Tcf4: T-cell factor 4; TUNEL: Terminal dUTP nick-end labelingThe authors declare that they have no competing interests.WW contributed to the major part of experimental work, interpreted the results, performed the statistics and drafted the manuscript. MC conceived the study, participated in its design and data analysis, and contributed with scientific discussion and manuscript preparation. SG contributed the normal hepatocytes and provided training and advice on culturing the hepatocytes. SKS is the principal investigator, responsible for conception of the project, designing the experiments, and approving the final manuscript. All authors read and approved the final manuscript.


When outcomes are binary, the c-statistic  is a standard measure of the predictive accuracy of a logistic regression model.An analytical expression was derived under the assumption that a continuous explanatory variable follows a normal distribution in those with and without the condition. We then conducted an extensive set of Monte Carlo simulations to examine whether the expressions derived under the assumption of binormality allowed for accurate prediction of the empirical c-statistic when the explanatory variable followed a normal distribution in the combined sample of those with and without the condition. We also examine the accuracy of the predicted c-statistic when the explanatory variable followed a gamma, log-normal or uniform distribution in combined sample of those with and without the condition.Under the assumption of binormality with equality of variances, the c-statistic follows a standard normal cumulative distribution function with dependence on the product of the standard deviation of the normal components (reflecting more heterogeneity) and the log-odds ratio (reflecting larger effects). Under the assumption of binormality with unequal variances, the c-statistic follows a standard normal cumulative distribution function with dependence on the standardized difference of the explanatory variable in those with and without the condition. In our Monte Carlo simulations, we found that these expressions allowed for reasonably accurate prediction of the empirical c-statistic when the distribution of the explanatory variable was normal, gamma, log-normal, and uniform in the entire sample of those with and without the condition.The discriminative ability of a continuous explanatory variable cannot be judged by its odds ratio alone, but always needs to be considered in relation to the heterogeneity of the population. Logistic regression models are frequently used to determine the association between a set of explanatory variables and a binary or dichotomous outcome variable. There are three primary reasons for fitting a logistic regression model: i) to determine the independent predictors of a binary outcome; ii) to determine the association between a specific variable and the probability of the occurrence of an outcome after adjusting for a set of other covariates; and iii) to predict the probability of the occurrence of a binary outcome given a specific vector of covariates. The third reason for fitting a logistic regression model occurs frequently in biomedical research, where researchers are interested in predicting the prognosis of individual patients.xy rank correlation between the predicted probability of the occurrence of the outcome and the observed outcome:Two key elements in assessing the performance of a fitted logistic regression model are the assessment of model calibration and model discrimination. Calibration refers to the agreement between observed outcomes and predictions, while discrimination refers to the ability of model predictions to discriminate between those with and those without the outcome,2. The dxy=2c−0.5.The discrimination of a logistic regression model can also be described by the area under the receiver operating characteristic (ROC) curve, often denoted by AUC. Each vaThe relationship between the c-statistic of a logistic regression model and the regression coefficients and the variance-covariance of the explanatory variables has not been fully explored. The objective of the current paper was to examine the relationship between the c-statistic and the regression parameters and the variance of the explanatory variable in the case of a univariate logistic regression model. We first use mathematical derivations to explicitly derive the relationship between the c-statistic and the outcome odds ratio and the variance of the continuous explanatory variable under the assumption that the continuous explanatory variable follows a normal distribution in subjects with and without the outcome. Second, we use Monte Carlo simulations to examine this relationship in a more general setting.Many derivations concerning the discrimination of different procedures require the assumption that the distribution of a continuous explanatory variable is normally distributed in those with the condition or outcome and also in those without the condition or outcome,6-9. ThuA and μU and variancesLet Y denote the dichotomous response variable indicating the presence or absence of the outcome or condition of interest. Let X denote the continuous explanatory variable. We assume that X has means μWe begin our derivation using a result derived by Zhou et al.. Using td denotes the standardized difference or Cohen’s effect size[where ect size-14. The 2 denote the common variance of explanatory variable in the two groups. Then, the log-odds ratio relating the explanatory variable X to the log-odds of the occurrence of the condition has the following property:In the special case when the explanatory variable has the same variance in those affected and unaffected by the condition, one can simplify the above result using a result from discriminant analysis. Let σβ=μA−μUσ2 .We used patients from the first phase of Enhanced Feedback for Effective Cardiac Treatment (EFFECT) Study, an initiative to improve the quality of care for patients with cardiovascular disease in Ontario,18. DetaThe outcome of interest for the current example was whether the patient died within 30 days of hospitalization. We fit two different logistic regression models. The first was a univariate logistic regression model in which we regressed 30-day mortality on patient age. In the second model, we regressed mortality on the following baseline covariates: age, sex, cardiogenic shock, acute congestive heart failure/pulmonary edema, systolic blood pressure, diastolic blood pressure, heart rate, respiratory rate, diabetes, hypertension, current smoking status, dyslipidemia, family history of coronary artery disease, cerebrovascular disease/transient ischemic attack, angina, cancer, dementia, peptic ulcer disease, previous AMI, asthma, depression, peripheral vascular disease, previous revascularization, congestive heart failure, hyperthyroidism, aortic stenosis, haemoglobin, white blood count, sodium, potassium, glucose, urea, and creatinine.We determined the empirical c-statistic for each of the two logistic regression models. We estimated the components of the distribution of age and of the linear predictor from the second logistic regression model that were necessary to predict the c-statistic using formulas (1) and (2). When using formula (2) with the multivariable model, we used β = 1, since the regression coefficient for the linear predictor would be one if the outcome were regressed on the linear predictor alone.The empirical c-statistic of the univariate logistic regression model that regressed 30-day mortality on age was 0.759. The corresponding predicted c-statistics were 0.760 and 0.790 when formulas (1) and (2) were used, respectively. The empirical c-statistic of the multivariable model was 0.853. The corresponding predicted c-statistics were 0.849 and 0.855 when formulas (1) and (2) were used, respectively. The improved accuracy of prediction of the c-statistic for the multivariable model is likely due to the distribution of the linear predictor having a distribution that is closer to a normal distribution compared to the distribution of age.X), such that the distribution of f(X) is normal in those with and without the condition.Under the assumption that the explanatory variable was normally distributed in those with and without the condition, we derived an explicit expression for the c-statistic. We demonstrated that the c-statistic is a function of only the mean and variance of the explanatory variable in those with and without the condition. In particular, the c-statistic is a function of the standardized difference comparing the mean of the explanatory variable between those with and without the covariate. When the explanatory variable had the same variance in those with and without the condition, we demonstrated that the model c-statistic is an increasing function of the standard deviation of the normal distributions and of the log-odds ratio. The primary novelty of our findings is that the functional relationship of the c-statistic of a logistic regression model has now been described. Using an extensive set of Monte Carlo simulations, we found that our formulas provided reasonably accurate prediction when the distribution of the explanatory variable was normal in the entire sample of those with and without the condition. Some of our findings corroborate previous observations based on Monte Carlo simulations on how the c-statistic improved with increases in the odds ratio. While oThere are two implications of these findings for researchers constructing and interpreting predictive models for binary outcomes. First, it is widely known that greater discrimination is possible when the regression model contains independent explanatory variables that are strongly associated with the outcome. HoweverIn conclusion, when a continuous explanatory variable is normally distributed both in those with and without the outcome or condition, and these two normal distributions have equal variances, then the c-statistic follows a standard normal cumulative distribution function with dependence on the product of the standard deviation of the normal components (reflecting more heterogeneity) and the log-odds ratio (reflecting larger effects). When the explanatory variable is normally distributed in the combined population of subjects, then the formulas that we derived provide a reasonably accurate prediction of the empirical c-statistic. We conclude that discriminative ability of an explanatory variable cannot be judged by its odds ratio alone, but always needs to be considered in relation to the heterogeneity of the population.The authors declare that they have no competing interests.PA derived the mathematical relationships, conducted the Monte Carlo simulations, and drafted the initial version of the manuscript. ES provided insight on study design, interpretation, and revised the manuscript for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2288/12/82/prepub


During infection, the virus exerts control over a multitude of host signaling pathways. Wnt/β-catenin signaling, an essential pathway involved in cell cycle control, differentiation, embryonic development, placentation and metastasis, is frequently dysregulated by viruses. How HCMV infection affects this critical pathway is not currently known. In this study, we demonstrate that HCMV dysregulates Wnt/β-catenin signaling in dermal fibroblasts and human placental extravillous trophoblasts. Infection inhibits Wnt-induced transcriptional activity of β-catenin and expression of β-catenin target genes in these cells. HCMV infection leads to β-catenin protein accumulation in a discrete juxtanuclear region. Levels of β-catenin in membrane-associated and cytosolic pools, as well as nuclear β-catenin, are reduced after infection; while transcription of the β-catenin gene is unchanged, suggesting enhanced degradation. Given the critical role of Wnt/β-catenin signaling in cellular processes, these findings represent a novel and important mechanism whereby HCMV disrupts normal cellular function.Human Cytomegalovirus (HCMV) is a ubiquitous herpesvirus that currently infects a large percentage of the world population. Although usually asymptomatic in healthy individuals, HCMV infection during pregnancy may cause spontaneous abortions, premature delivery, or permanent neurological disabilities in infants infected  A large percentage of the world population is infected with HCMV. As a leading viral cause of birth defects in the developed world, HCMV represents a significant public health burden. For the first time, we report that HCMV infection dysregulates the canonical Wnt/β-catenin signaling pathway which is essential in regulating a diverse range of biological functions. We demonstrate that HCMV infection leads to sequestration and degradation of β-catenin protein, the effector transcription factor in the pathway, thus preventing its downstream signaling activities. Since this pathway is essential in regulating mammalian development and homeostasis, the finding that HCMV impairs this pathway becomes globally important for understanding viral pathogenesis, particularly that related to HCMV disease. Human Cytomegalovirus (HCMV) is a betaherpesvirus that is ubiquitously present in the human population. Infection with HCMV is usually subclinical in healthy adults, but can cause serious disease in populations with underdeveloped or compromised immune systems Upon entry into the cell, HCMV manipulates the host environment in order to establish productive infection and ensure progression of the viral replication cycle. HCMV controls the host cell through dysregulating a multitude of signaling, cytoskeletal and regulatory pathways that control key cellular events. HCMV is known to highjack the host replication machinery One important signaling pathway that has been reported to be perturbed by herpesviruses is the canonical Wnt/β-catenin pathway  and metalloproteinase-9 (MMP-9)β-catenin, the major effector protein in the canonical Wnt signaling pathway, is normally retained in the cytoplasm in an inactive state through its interaction with a large protein complex comprised of axin, adenomatous polyposis coli (APC) and two serine/threonine kinases: glycogen synthase kinase-3β (GSK-3β) and casein kinase 1 (CK1). This complex maintains low levels of β-catenin in the cell through constant proteasome-mediated degradation. Phosphorylation of serine-45 by CK1 and subsequent phosphorylation on serine-33 and -37 by GSK-3β marks β-catenin for polyubiquitination and proteolytic degradation. Pathway activation is initiated when Wnt ligands, a large family of secreted glycoproteins, bind to heterodimeric Frizzled (FZD)/low-density lipoprotein receptor-related protein-5/6 (LRP-5/6) receptors on the surface of target cells. This initiates a cascade of events leading to phosphorylation and activation of Disheveled (Dsh/Dvl), a cytoplasmic scaffolding protein that relays Wnt signaling downstream and disrupts the axin/APC/GSK-3β complex. Disruption of the complex causes accumulation of stable, hypophosphorylated β-catenin in the cytoplasm, followed by its translocation to the nucleus  were transiently transfected with TOPflash or FOPflash containing mutated TCF/LEF-1 binding sites as a control. After 12 hr, transfected cells were infected with HCMV or mock-infected. At 48 hr after infection, the cells were stimulated with the canonical ligand Wnt-3A or lithium chloride (LiCl) for an additional 12 hr and then analyzed for luciferase expression. LiCl is a potent activator of Wnt signaling that acts downstream of Wnt receptors by inhibiting GSK-3β thus stabilizing β-catenin and promoting nuclear translocation cyclin D1, c-myc and Dikkopf-1 (DKK1) genes, which are transcriptionally regulated by the β-catenin/TCF/LEF-1 complex Next, the expression of endogenous Wnt/β-catenin target genes was examined in HCMV-infected cells. Expression of The effect of HCMV infection on the subcellular distribution of β-catenin in HCMV-infected HFFs was investigated by immunofluorescence. HFFs that had been infected with HCMV or mock-infected for 48 hr were immunostained for β-catenin . In mockTo determine if HCMV infection alters β-catenin protein levels, HFFs were infected with HCMV and analyzed for total β-catenin by Western blotting at various times after infection. There was a significant (up to 90%) reduction in the total levels of β-catenin protein in HCMV-infected HFFs by 72 hr after infection . Apart fβ-catenin levels are normally regulated by phosphorylation-dependent degradation via the ubiquitin-proteasome system de novo viral gene expression, HFFs were infected with HCMV or ultraviolet (UV)-inactivated HCMV. Immunofluorescence staining 24 hr after infection detected juxtanuclear accumulation of β-catenin in cells infected with HCMV virus, whereas in cells infected with UV-inactivated virions β-catenin failed to aggregate  cell line derived from first trimester placenta, into a migratory and invasive phenotype cyclin D1, MMP-2 and MMP-9, compared to mock-infected cells  is required to determine if they are indeed aggresomes.For β-catenin to drive transcription of target genes it must first translocate to the nucleus The mechanism(s) by which aggregated β-catenin is eliminated from the infected cells is not clear. Intracellular levels of β-catenin in the cell are maintained through GSK-3β mediated phosphorylation of β-catenin, which targets it for ubiquitination and degradation through the proteasome The HCMV viral particle consists of over forty structural and tegument proteins that are delivered to the host cell upon infection and are known to perturb multiple host signaling pathways The initial events taking place after HCMV infection are presumed to be essential for the establishment of virus replication and viral pathogenesis. HCMV is known to manipulate cell proliferation Dysregulation of Wnt/β-catenin signaling pathway by HCMV is likely to be of physiological significance particularly during congenital infection. It has been established that intrauterine transmission of HCMV during pregnancy is associated with abnormal placental development caused by impaired differentiation of EVTs. During gestation, these highly specialized cells differentiate into highly proliferative invasive cells that migrate through the decidua and remodel maternal spiral arteries to establish a vascular connection between the mother and the fetus cyclin D1 is required for initiation of S phase, after which the level of the protein drops. In this study, cyclin D1 expression that is dependent on β-catenin activation was modestly, but significantly downregulated in HCMV-infected SGHPL-4 cells and HFFs , a nuclear receptor that regulates lipogenesis and inflammation Invasion of EVTs during placental development involves production of extracellular matrix-degrading metalloproteinases and vasculogenic factors -4 cells . Consistβ-catenin is not only an essential component of canonical Wnt signaling, it is also an integral constituent of adherens junctions where it mediates contact between classical cadherins, α-catenin and the actin cytoskeleton. Our data indicate that membrane-associated β-catenin is also decreased in HCMV infected cells . Since tIn conclusion, we report for the first time that HCMV infection dysregulates the canonical Wnt/β-catenin signaling pathway in human dermal fibroblasts and placental EVTs. Our study establishes for the first time that HCMV inhibits canonical Wnt signaling by causing sequestration and degradation of endogenous β-catenin, thus preventing its downstream activities. Since the Wnt/β-catenin pathway is an evolutionarily conserved pathway involved in a diverse range of biological functions such as cell cycle control, cell differentiation, embryonic development, placentation and metastasis, the finding that HCMV impairs this pathway becomes globally important for understanding viral pathogenesis, particularly that related to HCMV disease.2. The human extravillous-like cytotrophoblast cell line SGHPL-4 cells was derived from first trimester chorionic villous tissue and exhibits features of invasive cytotrophoblasts, such as expression of HLA-G, CD9 and cytokeratin-7 2. All experiments were carried out using either a laboratory strain of HCMV that expresses GFP from the UL127 promoter (Towne-GFP), or the HCMV BAC-derived clinical strain TR, both kindly provided by Dr. Dan Streblow . All viral strains were propagated in HFFs. For HCMV infections, cells were synchronized by serum starving overnight and infected with HCMV at multiplicity of infection (MOI) of 1 to 2. Briefly, viral inoculum was added to the cells and allowed to adsorb for 90 min at 37°C in 5% CO2. The virus inoculum was then removed and replaced with fresh medium containing 0.5% FBS.Human foreskin fibroblasts (HFFs) were purchased from American Type Culture Collection  and were cultured in Dulbecco's Modified Eagle Medium  supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin-L-glutamine at 37°C in 5% COPrimary antibodies: mouse anti-β-catenin , mouse anti-β-actin , mouse anti-CMV IE1/2 , mouse anti-CMV p65 , mouse anti-γ-tubulin , rabbit anti Histone 4 (H4) , rabbit anti-GAPDH , rabbit anti-caveolin-1 . Secondary antibodies: AlexaFluor antibodies were purchased from Invitrogen and used at 1∶1000 dilution: AlexaFluor 488 goat anti-mouse, AlexaFluor 555 goat anti-mouse. Human recombinant Wnt-3A was purchased from R&D . Lithium chloride (LiCl) was purchased from Sigma-Aldrich.4Cl solution in DPBS for 20 min followed by permeablization in 0.1% Triton X-100 for 8 min. Prior to blocking, the cells were incubated with an Fc receptor blocker  for 30 min at room temperature. The cells were then blocked in 5% bovine serum albumin (BSA) for 1 hr at room temperature. After blocking, cells were incubated with primary antibodies diluted in blocking solution at the manufacturer's recommended dilution for 1 hr at room temperature. The cells were washed and incubated for 1 hr with AlexaFluor-conjugated secondary antibodies and 4′-6-Diamidino-2-phenylindole (DAPI) as a nuclear counterstain. Coverslips were washed and mounted with ProLong gold antifade reagent (Invitrogen) and imaged at 40× using a Zeiss Axioplan II microscope . Processing of the acquired images was performed using Adobe Photoshop software.For all immunofluorescence analyses, cells were seeded onto 1.5 mm glass coverslips coated with 0.2% gelatin, and infected with HCMV or mock-infected as described above. At indicated time points after infection, cells were washed with Dulbecco's phosphate-buffered saline  and fixed in 2% paraformaldehyde  for 20 min. Cells were washed with DPBS and incubated with 50 mM NHTo evaluate activation of the Wnt/β-catenin pathway, SGHPL-4 cells were transiently transfected with the TCF/LEF-1 reporter plasmid TOPflash, which contains multimeric TCF/LEF-1 sequences upstream of a firefly luciferase reporter gene. FOPflash plasmid containing mutated TCF/LEF-1 binding sites was used as a specificity control for TOPflash activity. Both TOPflash and FOPflash plasmids were obtained from Millipore . A Renilla luciferase-expressing plasmid pRL-TK  was co-transfected with both TOPflash and FOPflash to serve as an internal transfection control. For each transfection, SGHPL-4 cells were transfected with 1 µg of either TOPflash or FOPflash DNA and 0.1 µg of Renilla plasmid using the Neon transfection system (Invitrogen) according to the manufacturer's instructions . Each transfection was performed in triplicate. Six hr after transfection, the cells were infected with HCMV (MOI of 1 to 2) or mock-infected. Forty-eight hr after infection, the cells were stimulated with 150 ng/ml Wnt-3A or PBS vehicle control for an additional 12 hr and cells were harvested. Luciferase activity was assayed with a dual-luciferase reporter assay kit  and measured by a Lumat LB 9507 tube luminometer . For each sample, the firefly luminescence signal was normalized to the corresponding Renilla signal.4 cells per insert. Wnt-3A ligand (150 ng/ml) or PBS control was added to both the upper and the lower chambers of the multiwell plate. Each condition was performed in triplicate. The cells were allowed to migrate through the FluoroBlok membrane for 12 hr at 37°C in 5% CO2, after which they were fluorescently labeled with calcein AM  and visualized by fluorescent microscopy. The 12 hr timepoint was empirically determined to be optimal for SGHPL-4 cell migration. Three random fields from each insert were captured with a Nikon TE200 inverted fluorescent microscope  and the average fluorescence intensity was determined by ImageJ analysis software.SGHPL-4 cells were infected with HCMV or mock-infected at an MOI of 1 to 2. Cells were trypsinized 48 hr after infection, collected in serum-free F-10 media and added to the upper side of an 8 µm FluoroBlok 24-well multiwell insert system  at a density of 5×10HCMV was UV-inactivated by exposure to 426 mJ of 254 nm UV light in Bio-Rad GS gene linker UV Chamber . UV inactivation was assured by the absence of HCMV IE1/2 expression in HFFs.5′-CGCCCTCGGTGTCCTACTTC-3′), cyclin D1 reverse (5′-GACCTCCTCCTCGCACTTCTG-3′); MMP-2 forward (5′-ATGTCGCCCCCAAAACGGACAAAG-3′), MMP-2 reverse (5′-CGCATGGTCTCGATGGTATTCTGG-3′); MMP-9 forward (5′-AGACGGGTATCCCTTCGACG-3′), MMP-9 reverse (5′-AAACCGAGTTGGAACCACGAC-3′); β-catenin forward (5′-T ACAAACTGTTTTGAAAATCCA-3′), β-catenin reverse (5′-CGAGTCATTGCATACTGTCC-3′); DKK1 forward (5′-TTCCAACGCTATCAACCTGC-3′), DKK1 reverse (5′- CAAGGTGGTTCTTCTGGAATACC-3′); c-myc forward (5′- GCCACGTCTCCACACATCAG-3), c-myc reverse (5′-TCTTGGCAGGAGGATAGTCCTT-3′); 36B4 forward (5′-TGGAGACGGATTACACCTTC-3′), 36B4 reverse (5′-CTTCCTTGGCTTCAACCTTAG-3′); Human GAPDH mRNA was measured using a TaqMan Gene Expression Assay according to the manufacturer's protocol . Prior to performing real-time PCR on experimental samples, primer concentrations were optimized to provide equal priming efficiency (∼100%) for each primer pair. Negative controls, including cDNA reactions without reverse transcriptase or RNA and PCR mixtures lacking cDNA were included in each PCR. Following amplification, specificity of the reaction was confirmed by melt curve analysis. Relative quantitation was determined using the comparative CT method with data normalized to GAPDH or 36B4 and calibrated to the average ΔCT of mock-infected control at the specified time point.Total RNA was collected from mock and HCMV-infected cells using the Qiagen RNeasy Kit  according to the manufacturer's instructions. RNA (500 ng) was reverse transcribed using Bio-Rad iScript cDNA synthesis kit (Bio-Rad) and PCR reactions were performed using SYBR Green supermix (Bio-Rad) or TaqMan Universal PCR  master mix and the iCycler Real-Time PCR detection system (Bio-Rad). Oligonucleotide primers  used are as follows: cyclin D1 forward  supplemented with protease inhibitor cocktail . The lysates were sonicated briefly and protein content was determined by Bradford assay (Bio-Rad). Equivalent amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose. The blots were blocked with 5% BSA and incubated overnight with primary antibodies diluted in blocking solution at the recommended dilutions at 4°C. Following three washes with Tris-buffered saline (TBS), blots were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies (Invitrogen) diluted in 5% BSA at 1∶10,000. After washing in TBS, proteins were detected using SuperSignal chemiluminescent substrate  according to the manufacturer's instructions. For densitometric analysis of Western blot images, density profiles of the bands were measured using ImageJ software.Membrane/cytoskeletal, cytoplasmic and nuclear fractions were isolated from HFFs that had been mock and HCMV-infected for 48 hr, using the Qproteome cell compartment kit (Qiagen) according to the manufacturer's protocol. Resulting fractions were separated on SDS-PAGE and immunoblotted for β-catenin. Histone H4, GAPDH and caveolin-1 were used to assess the purity of the cell fractions as previously described 4) were plated in a 96-well tissue culture plate in 100 ìl DMEM supplemented with 10% FBS. The cells were infected with HCMV-TR (MOI of 1 to 2) or mock-infected. Cells were analyzed at 24, 48 and 72 hr after infection using a Proteasome Assay kit  that measures the catalytic activity of the 26S proteasome, according to the manufacturer's instructions. The fluorescence intensity was measured at an excitation of 360 nm and emission of 480 nm using a fluorescence plate reader.HFF cells (1.5×10t test using GraphPad Prism 4 software. Data are presented as the means ± standard error of the means (SEM).Data from HCMV-infected groups were compared to mock-infected groups and significant differences were determined by Student's t-test or one-way analysis of variance (ANOVA) followed by Tukey's post hoc


The arginine methyltransferase PRMT5-MEP50 is required for embryogenesis and is misregulated in many cancers. PRMT5 targets a wide variety of substrates, including histone proteins involved in specifying an epigenetic code. However, the mechanism by which PRMT5 utilizes MEP50 to discriminate substrates and to specifically methylate target arginines is unclear. To test a model in which MEP50 is critical for substrate recognition and orientation, we determined the crystal structure of Xenopus laevis PRMT5-MEP50 complexed with S-adenosylhomocysteine (SAH). PRMT5-MEP50 forms an unusual tetramer of heterodimers with substantial surface negative charge. MEP50 is required for PRMT5-catalyzed histone H2A and H4 methyltransferase activity and binds substrates independently. The PRMT5 catalytic site is oriented towards the cross-dimer paired MEP50. Histone peptide arrays and solution assays demonstrate that PRMT5-MEP50 activity is inhibited by substrate phosphorylation and enhanced by substrate acetylation. Electron microscopy and reconstruction showed substrate centered on MEP50. These data support a mechanism in which MEP50 binds substrate and stimulates PRMT5 activity modulated by substrate post-translational modifications. PRMT5-MEP50 forms an unusual tetramer of heterodimers with four copies each of PRMT5 and MEP50. We demonstrate that PRMT5-MEP50 activity is modulated by substrate post-translational modifications and that MEP50 is required for stimulating PRMT5 activity. Our data suggest that a primary function of MEP50 is to bind and orient the arginine-containing substrate to the PRMT5 catalytic site. Furthermore, PRMT5 enzyme turnover may be modulated by charge-shifting substrate post-translational modifications.Here we report the structure of the full-length While this manuscript was under review, the highly similar human PRMT5-MEP50 structure was independently reported Xenopus PRMT5-MEP50 complex in the presence of SAH (D2 symmetry). Crystallographic symmetry results in an XlPRMT5 dimer similar to those observed in all reported PRMT structures, including CePRMT5  is similar to CePRMT5 (PDB:3UA3). Discrete N- and C-terminal domains are connected by an unstructured loop  of 1.1 Å . The long dimerization arm in CePRMT5 is a small loop in the Xenopus structure . We observed interpretable electron density for a segment connecting the N-terminal TIM barrel and the C-terminal Rossmann fold of XlPRMT5. Therefore our assignment of the orientation of the arrangement of the N-to-C terminal domains differed from that assigned to the C. elegans PRMT5 (PDB:3UA3). Sequences of Xenopus and human PRMT5 N-terminal domains are highly similar with only minor amino acid changes  clearly showed bound SAH . The prominent electron density assigned to the SAH sulfur in the Xenopus structure allows for confident placement adjacent to a small channel that connects the PRMT5 catalytic site with bulk solvent. This model suggests that the methyl donor on SAM would also be facing solvent and therefore this channel could support the entry of the substrate arginine guanidinium group into the catalytic site. This narrow catalytic site entry pocket on the outer face of PRMT5 is adjacent to the N-terminus of its dimer-paired PRMT5 and MEP50, and distal to its directly-associated MEP50 (C. elegans PRMT5 structure (αA helix in 3UA4).We readily identified SAH in a conserved catalytic site. Difference Fourier synthesis (Found SAH . The relHsPRMT5 is shown (The invariant glutamic acid residues (Glu431 and Glu440) in the “double-E” loop are hydrogen bonded to SAH . The PRMMEP50 adopts a seven-bladed toroidal WD40 repeat . The lasXenopus). PRMT5 is also an acidic protein with a theoretical pI of 5.8. The electrostatic surface potential of the PRMT5-MEP50 complex reveals an extended negatively charged surface, consistent with recruitment of basic substrates such as histone tails (We compared MEP50 with WDR5 (PDB:2H68), a WD-repeat protein that recognizes histone H3 tails for the MLL lysine methyltransferase ne tails .XlPRMT5-MEP50 assembly of 405.4 kDa, also consistent with a tetrameric complex . Experiments performed in 2 M NaCl exhibited no change in mass, consistent with a stable tetramer. Sedimentation velocity experiments revealed sedimentation and diffusion coefficients consistent with both the equilibrium value as well as the Hydropro  complex . FinallyThere are numerous points of interaction within the PRMT5 dimer pair and the tetramer interface, including contributions from the N-terminal TIM barrel domain as well as in the Rossmann fold and β-barrel in the C-terminus  greatly reduced the activity of PRMT5-MEP50. The enzyme also methylated the known substrate Nucleoplasmin C-terminal tail peptide and the histone H2A.X-F N-terminal, but not C-terminal peptide . Human PRMT5 (Flag-HsPRMT5), 84% identical to the Xenopus protein (X. laevis MEP50 (XlMEP50), we co-purified the proteins on anti-Flag resin. XlMEP50 specifically eluted with Flag-HsPRMT5 . We imaged XlPRMT5-MEP50 complex and found a novel arrangement of PRMT5-MEP50 dimers that assembles to form a tetramer. Our data conclusively show that XlPRMT5 forms a homotetramer in the crystalline state and in solution. The residues that make intra-tetramer contacts are 100% conserved between Xenopus, human and cattle PRMT5 and only have a single R to H change with mouse and rat PRMT5. There is no conservation in these residues between Xenopus PRMT5 and Arabidopsis, Drosophila, C. elegans PRMT5 or S. cerevisiae Hsl7. Superposition of the C. elegans structure and the Xenopus structure showed that the extensive loop found in the dimerization arm (residues 551 to 586 in CePRMT5 compared to residue 483 to 491 in XlPRMT5) is incompatible with tetramerization as it would clash with the tetramer-paired PRMT5. These observations suggest that if non-vertebrate PRMT5 forms higher order assemblies it will be through a mechanism distinct from that responsible for the tetramer formation observed in our studies.We determined the crystal structure of the full-length SAH–the product of the methyltransferase reaction–is substantially buried, implying that the SAM methyl donor is possibly accessible to substrate through a modest channel that connects the catalytic site with bulk solvent. Each active site is oriented in line with the cross-dimer paired MEP50. We showed that MEP50 is required for significant PRMT5 methyltransferase activity. High-density histone peptide arrays and solution assays demonstrated that the substrate charge-modulating post-translational modifications of lysine acetylation and serine phosphorylation stimulated or inhibited PRMT-MEP50 activity, respectively.The function of the tetramer is unclear, and many residues will need to be mutated to disrupt the complex to test its function. The tetramer may serve to promote processive methylation of neighboring substrates (such as in a Nucleoplasmin:histone complex or on chromatin). PRMT5 has been observed interacting with many proteins in addition to MEP50. Therefore, the tetramer may act as a landing-pad for other proteins – such as pICln, RioK1 and CoPR5– that are known interactors and modulators of PRMT5 activity All PRMTs published to date form dimers or higher order structures. A long-standing issue has been the functional and mechanistic basis for this dimerization. One possibility would be to enhance processive dimethylation by allowing two neighboring active sites to function together by successive turnover on a bound substrate. Our previous study showed that only increased concentration of PRMT5-MEP50 promoted dimethylation, consistent with a distributive catalysis mechanism et al. Xenopus PRMT5 and MEP50 are highly identical, there is no obvious MEP50 ortholog in C. elegans. The low sequence identity  between XlPRMT5 and CePRMT5 suggests that CePRMT5 may interact with substrate using a different mechanism.PRMT5 typically coexists with MEP50 and the functional nature of this complex has long been questioned. MEP50 has been previously shown to interact with histone H2A RGG or GRGK motifs)  . The posXenopus PRMT5-MEP50 structure we observed that these invariant glutamic acid residues (Glu431 and Glu440) are hydrogen bonded to SAH, in contrast to the C. elegans and human structures . However, our analysis shows an alternative channel, exposed to bulk solvent, which would permit arginine guanidinium entry in line with the SAH sulfur and the two catalytically important glutamic acid residues. Intriguingly, these invariant glutamic acid residues (Glu431 and Glu440) in the “double-E” loop are hydrogen bonded to aminoethanoic acid of homocysteine in our model. Our model may provide another mode for PRMT5 substrate interaction and would allow peptide interact with the dimer-paired MEP50  and a SAM analog rather than SAH We observed inhibition of PRMT5 activity when the substrate contained Ser1 phosphorylation. We suggest that this charge-shifting PTM may displace the unstructured substrate tail and displace the arginine, two amino acids away, from the active site. Interestingly, this modification is enriched in mitotic and S-phase cells HsPRMT5 enzyme was purchased from SinoBiologicals . HeLa octamers and mononucleosomes were purified as described after growth in the presence or absence of 40 mM Na Butyrate for 16 hours Xenopus PRMT5 and MEP50 were produced as described Chemicals and reagents were obtained from Sigma  or Fisher Scientific . Sf9 and High Five cells were grown in Sf-900 III with L-glutamine and Express Five Medium , respectively, with penicillin and streptomycin. Recombinant Flag-6 cells/ml culture with baculovirus while shaking at 27°C for 60 hours PRMT5 and MEP50 proteins were co-expressed in Hi5 cells by infecting 500 ml of 2×10XlPRMT5-MEP50 protein complexes were co-expressed in Hi5 cells with baculovirus in ESF-921 medium without methionine while shaking at 27°C. 100 mg/L of D,L-Selenomethionine was added to the medium 20 h post-infection and an additional 50 mg/L of D,L-selenomethionine was added 44 h post-infection. The cells were harvested 71 hour post-infection and purified.Hi5 cells were adapted to ESF-921 medium without methionine (Expression Systems). XlPRMT5-MEP50-SAH complexes were prepared by incubating ∼8–10 mg/ml XlPRMT5-MEP50 in 400–500 mM NaCl, 10% glycerol, 5 mM DTT and 20 mM ADA pH 6.5 on ice with 1 mM SAH. The complex was co-crystallized in 30%–40% MPD and 100 mM Bis-Tris pH 5.5 at 18°C using hanging drop or sitting drop vapor diffusion methods. Crystals were directly flash frozen in liquid nitrogen before data collection. X-ray diffraction data were collected at the X25 beamline of Brookhaven National Laboratory on a Pilatus 6 M detector at 100° K. The data were processed with the HKL2000 program suite XlPRMT5 was calculated based on the structure of CePRMT5 . For the equilibrium experiments, complex at three concentrations  in 20 mM ADA, 250 mM NaCl, 10% (v/v) glycerol and 5 mM DTT at pH 6.5 were loaded into the six channel cells. The samples were sequentially equilibrated at rotor speeds of 5,000 and 9,000 rpm for 24 hr each at 20°C in a Ti-60 rotor. The sedimentation equilibrium data were analyzed using version 1.1.44 of HeteroAnalysis Sedimentation equilibrium and velocity experiments with the PRMT5-MEP50 complex protein were performed with a Beckman XL-I analytical ultracentrifuge using the absorption optics set to 280 nm. The buffer density and the partial specific volume of the complex were calculated using the Sednterp software (−1. The details of the FPLC-SAXS experiment at BL4-2 were described previously SAXS was performed using Bio-SAXS beam line BL4-2 at Stanford Synchrotron Radiation Lightsource (SSRL) http://ssrl.slac.stanford.edu/~saxs/analysis/sastool.htm) was used for scaling, azimuthal integration and averaging of individual scattering images after inspection for any variations potentially caused by radiation damage. The first 100 images were scaled and averaged to create a buffer-scattering profile, and this was then subtracted from each of the subsequent images to produce the final scattering curve for each exposure. The 9 scattering profiles over an elution peak were averaged and then used for curve fitting with crystal structure using the program CRYSOL The data acquisition program Blu-ICE 15 µg of PRMT5 and MEP50 in 20 mM ADA pH 6.5, 250 mM NaCl, 10% glycerol, and 5 mM DTT was subjected to size exclusion chromatography using a WTC030N5 (Wyatt Technology Corporation) column coupled to a Shimadzu HPLC system. Light scattering measurements were performed downstream, using a miniDawn TREOS instrument connected to the column output, followed by Optilab rEX refractive index analysis (Wyatt Technology Corporation). Control experiments were carried out with BSA diluted in the same buffer as the sample buffer. Data from these experiments was collected and interpreted using Astra software (version 6.0.3.16).2HPO4, 1.47 mM KH2PO4, 1 mM PMSF, pH 7.4) for 3 hours at room temperature. After washing the beads three times with 500 µl PBS/0.1% Tween, they were incubated with 100 nM recombinant protein in PBS over night at 4°C. The next day the beads were washed four times with 500 µl Buffer D  and once with 500 µl Lower Hepes . Finally the beads were resuspended in 50 µl Tris  and 10 µl 6x loading dye and boiled at 95°C. The protein samples were separated via SDS PAGE together with an input control (5%) followed by western blot analysis.5 µg biotinylated peptides were incubated with 20 µl magnetic streptavidin-coupled beads  in PBS  were coupled to anti-MEP50 antibodies or pre-immune serum (as control) for 1.5 hours in HNTG buffer . After washing the beads, 50 µl egg extract (HSS) were incubated with the first half of the antibody-coupled beads for one hour, followed by a second round of depletion with the other half. The MEP50-depleted egg extract was used for further analysis via western blot and methyltransferase activity assay.2, 2 mM EGTA, 0.1% Triton X-100, 0.5 mM PMSF) for one hour at 4°C.Nuclei were isolated from HeLa cells and MNase digested in TM2 buffer 3H-SAM in 15 µl reaction buffer  for 20 min at 30°C. The reconstituted Flag-HsPRMT5-XlMEP50 was pre-incubated in equimolar amounts for 15 min at room temperature prior to the reaction. The reaction mix was spotted on P81 filter paper, washed with sodium carbonate buffer , air-dried and analyzed via scintillation counter .Histone 1–20 peptides  or Npm peptides  at 3.3 µM final concentration, or 0.5 µg full-length histones or histone complexes, were incubated with 50–220 nM recombinant PRMT5-MEP50 and 0.5 µM Xenopus MEP50 and Flag-HsPRMT5 in TBS at 4°C. As a negative control, MEP50 was incubated under the same conditions without PRMT5. After 2 hours, the suspension was transferred to a Mini-spin column and centrifuged . After washing, the proteins were eluted from the beads and analyzed via western blot (α-PRMT5 and α-MEP50 antibodies).20 µl Anti-Flag M2 antibody-coupled agarose beads were incubated with equimolar amounts (50 nM) of www.jpt.com). Peptides synthesized using spot synthesis www.r-project.org).A library of 20-mer peptides spanning the sequences of histones H2A (P0C0S8), H2B (P62807), H3 (P68431) and H4 (P62805) was generated including single known modifications and in various combinations (sequences available at http://www.cgl.ucsf.edu/chimera) and then refined with automatic fitting package Situs http://www.emdatabank.org).PRMT5-MEP50 alone or in complex with Npm (at a molar ratio of 1∶2) were applied to carbon-coated copper grids and negatively stained for ∼30 seconds with 2% uranyl acetate. Micrographs viewed on a JEOL JEM-2100F electron microscope were recorded on a TVIPS TemCam-415 CCD camera with a pixel size of 1.67A at specimen space. Images of individual of PRMT5-MEP50 were boxed out and classified into homogeneous classes using multivariant statistical analysis and the selected class averages were used to build initial three-dimensional density model. An alternative density model was computed from those images with strong C2 symmetry and the images with strong mirror symmetry. Refinement was carried out by confronting data images with calculated projections from the density models using methods implemented as previously described Figure S1PRMT5 and MEP50 domain organization and structural homology.A.XlPRMT5 domain organization, with the top plot highlighting the major subdomains, including the TIM barrel, Rossmann fold (location of nucleotide binding) and a β-barrel fold. The interacting residues determined in the structure are shown on top. The lower half shows the discrete structural domains and the flexible connector loop between the N- and C-terminal units. The inset boxes show the Xenopus N- and C-terminal domains overlaid over the CePRMT5 structure (3UA3). B.XlMEP50 organization, with the top plot indicating the location of the “cross-dimer insertion loop” (yellow) and the residues that form explicit contacts with PRMT5 (green). The lower part shows the structure and highlights the insertion loop in yellow. The inset shows MEP50 overlaid with WDR5 (2H9M). The location of residues missing from the MEP50 structure is indicated. C. An electrostatic potential map of XlPRMT5-MEP50 is plotted from 3 different perspectives, as indicated, with red surfaces acidic and blue surfaces basic.(PDF)Click here for additional data file.Figure S2PRMT5 conservation across evolution and alignment.A. PRMT5 amino acid identity was calculated using the MAFFT alignment in Geneious v5.5. B. A PRMT5 multiple sequence alignment (without S. cerevisiae Hsl7) is shown with conserved residues positions highlighted in black and divergent residues in white. Locations of interaction domains determined in the structure are highlighted above the plot.(PDF)Click here for additional data file.Figure S3The SAH-omitted electron density map near the SAH binding site. The SAH molecule and some surrounding residues are drawn in gray and yellow color, respectively. The mFo-Fc map (difference map) at 3 σ is shown in green and the 2Fo-Fc map at 1.5 σ is shown in blue.(PDF)Click here for additional data file.Figure S4PRMT5 interacting residues with SAH, dimer interface, tetramer interface, and with MEP50. A. Residues that we identified in the structure interacting with SAH are shown, with their corresponding hydrogen bonded atom in SAH and the distance in angstroms. *indicates residues that may also be involved in catalysis. B. Ligplot representation of the hydrogen bonding and neighboring residues around SAH in the structure. Inset: SAH pose from 3UA3. C. PRMT5 dimer interface residues, split into salt bridges in the dimerization arm and salt bridges and hydrogen bonds in the N- and C- terminal domains. D. PRMT5 tetramer interface residues, split into salt bridges and hydrogen bonds, with distances listed in angstroms. E. PRMT5 and MEP50 interacting residues are shown, with salt bridges, cation-Π interactions and hydrogen bonds illustrated. F. Cartoon representation of the PRMT5 152–178 loop and its interactions with MEP50.(PDF)Click here for additional data file.Figure S5R3me2s antibody response on peptide array. A high-density peptide array was probed with R3me2s antibody (Millipore #07–947) to measure the baseline signal to determine if neighboring PTMs modulate the response. Peptides are listed in text on the left. In the middle, black boxes represent the presence of a particular modification on a peptide. The histogram on the right shows the relative antibody signal. Pink bar shows the signal on the H4(1–20)R3me2s peptide. Green boxes show the presence of R3me1 or R3me2s.(PDF)Click here for additional data file.Figure S6Recombinant human PRMT5 is not complexed with MEP50.A. Recombinant human PRMT5 produced in 293 cells was immunoblotted for PRMT5 . B. Recombinant GST-tagged human MEP50 and human PRMT5 were immunoblotted with an antibody specific for human MEP50 .(PDF)Click here for additional data file.Figure S7Electron microscopy and reconstruction.A. Recombinant class average 2D projections of PRMT5-MEP50. B. PRMT5-MEP50 incubated with recombinant Nucleoplasmin class average 2D projections. The additional density from Nucleoplasmin was observed centered on MEP50 (yellow arrows). C. 3D electron microscopy reconstruction of PRMT5-MEP50 complexed with Nucleoplasmin. PRMT5-MEP50 and Nucleoplasmin molecules from the structure were placed in the density map. The density map is shown wire mesh.(PDF)Click here for additional data file.Figure S8SAH Pose Comparison. A. SAH in Xenopus PRMT5 (PDB:4G56). H-bonds are indicated as dashed yellow lines. The sulfur atom is shown in yellow. B. SAH in human PRMT5 (PDB:4GQB), H4R3 in green, PRMT5 in pink. C. SAH of Xenopus and human PRMT5 overlaid. The aminoethanoic acid in the Xenopus structure clashes with the H4R3 guanidinium position in the human structure. D. SAH of rat PRMT1 , human PRMT3 , mouse PRMT4 , C. elegans PRMT5 , and human PRMT5 .(PDF)Click here for additional data file.


By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust β-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent β-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of β-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2Kb promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63+ cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling  Wnt signaling exerts diverse biological effects in an organism, such as body axis determination during embryogenesis, cell polarity induction and stem cell self-renewal Association of Wnt ligand to its receptor/co-receptor complex initiates canonical Wnt signaling. In the absence of Wnt ligands, the β-catenin “destruction complex” comprising Axin, adenomatosis polyposis coli (APC) and GSK-3β, sequesters and phosphorylates the canonical Wnt signal transducer, β-catenin. Phosphorylated β-catenin is targeted for ubiquitination and proteasomal degradation. Hence, in the unliganded state, the cytoplasmic pool of β-catenin is maintained at low levels. Upon association of a Wnt ligand to an appropriate Fz receptor and co-receptor LRP5/6, the regulatory protein Dishevelled (Dvl) becomes activated via phosphorylation. Activated Dvl triggers disruption of the destruction complex, allowing for nuclear accumulation of β-catenin, where it interacts with the LEF/TCF complex, resulting in the transcription of Wnt target genes.in vitro experiments have many practical benefits, such as typically shorter duration and high throughput data analysis, the complexity of multi-lineage systems attainable in genetically engineered mouse (GEM) models often provides a more physiologically relevant understanding of a particular biological process. Since the technique was developed in the early 1980s, many transgenic mouse models have been characterized. Manipulation of regulatory pathways, via selective gene regulation in transgenic models, has provided important insights into tumor development and cancer biology. More recent “conditional” technologies, such as Cre-Lox recombination or tamoxifen-inducible transcriptional regulation, have added temporal control of in vivo gene manipulation. These systems have proven to be especially useful in models where alteration of a biological pathway may result in embryonic lethality. However, reversible regulation of gene expression or protein function can provide an additional dimension of control to permanent Cre-Lox-induced changes.Due mostly to lipid modifications, purification of Wnt proteins has proven to be technically challenging. Therefore, in order to reproduce Wnt activation in experimental settings, various strategies have been developed to selectively stabilize or upregulate β-catenin expression. Although 580S/580S mouse model (APCflox/flox) promoted adenoma formation in the colorectal region by 4 weeks of age + (PB-Cre+) mice were crossed with APCflox/flox to generate PB-Cre+; APCflox/flox. These mice allow for the inactivation of APC specifically within prostate epithelial cells, leading to development of prostate adenocarcinoma within 7 months The importance of Wnt signaling in tumor biology has led to a keen interest in dissecting and identifying key regulatory components. The mouse mammary tumor virus (MMTV)-based tumor model provided initial insight into the importance of Wnt signaling in the regulation of tissue homeostasis. Int-1 (Wnt1) was the first gene discovered to be activated by an integrated provirus in the MMTV tumor models A second method for conditional activation of Wnt signaling involves stabilization of β-catenin by excising a portion of exon 3, the target of phosphorylation and subsequent ubiquitination and degradation. Transgenic mice expressing conditionally activated β-catenin in neural precursors develop enlarged brains and increased cerebral cortical surface area v). Like a “true” switch, iLRP5 has the ability to be turned on and off by the simple addition or removal of CID, AP20187, resulting in potent canonical Wnt pathway induction.Considering the prevalence of Wnt signaling-based mouse models that are constitutively active, we opted to develop an inducible canonical Wnt signaling system that utilizes the more versatile chemical inducer of dimerization (CID)-based “on/off” system. Toward this goal, we performed CID-based oligomerization of several components of Wnt signaling and discovered that inducible multimerization of the cytoplasmic domains of the Wnt coreceptors, LRP5 and LRP6, could emulate canonical Wnt signaling, without the potentially confounding effects of LRP5/6 interaction with Wnt inhibitors, Dickkopf proteins (DKKs) and sclerostin (SOST) b) to express iLRP5 and a coupled click beetle (species) red-shifted luciferase reporter (CBR) gene. This allowed for the near ubiquitous expression of iLRP5, as well as bioluminescence imaging in live tissues. Initially, we have analyzed the consequence of Wnt activation via iLRP5 dimerization in mammary and prostate tissue systems. Both of these systems exhibited highly reproducible CID-dependent hyperplasia and in some instances tumorigenesis. These transgenic mice, named Ubiquitous Activator of β-catenin (or “Ubi-Cat”), should, therefore, provide a valuable research tool for controlled induction of the Wnt pathway in various tissues.To better understand the disparate outcomes of ß-catenin induction in physiological settings, we used the broadly active major histocompatibility complex I (MHC-I) promoter (H-2KF-LRP5c and MF-LRP6c) constitutively activates β-catenin signaling in mammalian cells, suggesting that membrane localization of the signaling domain of LRP5/LRP6 is a major determinant for β-catenin induction Fig. S1). Therefore, we reasoned inducible recruitment of the cytoplasmic domain of LRP5/LRP6 to membrane lipid rafts might result in reversible regulation of signaling. To test this, we fused the LRP5 cytoplasmic domain to three tandem rapamycin-binding FKBP12 domains F) to tandem copies of the mutant, 89-amino acid, rapamycin-binding domain from mTOR /FRBl) F-FRBl2, β-catenin signaling is induced, as determined using the TCF4/β-catenin-responsive reporter, TCF-SEAP (left panels). However, the signaling level was lower than permanently lipid raft-“anchored” LRP5 (MF-LRP5c), suggesting that membrane translocation alone is not sufficient for maximal β-catenin induction.Consistent with plasma membrane-anchored LRP5/LRP6 variants, lipid raft targeting of the intracellular domains of LRP5 and LRP6 . Indeed, in addition to low-level β-catenin induction achieved by membrane localization, further dimerization (or oligomerization) of LRP5 cytoplasmic domains, via dimeric FK506, FK1012 , bottom right), suggesting that synthetic dimerization of LRP5 recapitulates the reported functional role of LRP5/6 clustering and activation During lipid raft targeting, the high local concentration of proteins that occurs likely leads to increased incidental protein aggregation that could contribute to activation of signaling. Therefore we tested the hypothesis that further regulated oligomerization of membrane-targeted F3-LRP5c might further enhance β-catenin activation (F-FRBl2), led to robust β-catenin induction, indistinguishable to that achieved by dimerization (or oligomerization) of membrane-localized F3-LRP5c (v”), that responds to FKBP12v36 (Fv)-selective CID, AP20187  Several reports have shown that LRP5 phosphorylation by membrane-associated kinases, such as CK1γ and GSK3β, is required for Axin sequestration and induction of canonical Wnt signaling, again suggesting membrane targeting of LRP5 is critical F-LRP5c or iLRP5 completely abrogated both MF-LRP5c- and iLRP5-mediated β-catenin activation, respectively (S2). In addition, dimerization of cytoplasmic iLRP5 induced the interaction of dimerized iLRP5 and Axin, suggesting that Axin is a critical downstream target of endogenous LRP5, as well as iLRP5 (It has been well documented that sequestration of Axin from the destruction complex by membrane-bounded LRP5 is a key step for β-catenin induction (16). Therefore, we tested whether interaction with Axin by dimerized iLRP5 is required for β-catenin activation. Co-expression of Axin with Mas iLRP5 Fig. 2C.Although dimerized F3-LRP5c could stabilize β-catenin without direct membrane localization, we could not rule out that dimerization of LRP5c had conferred increased membrane affinity, possibly due to Fz/Dvl complexes, consistent with the putative role for Dvl in LRP6 aggregation in the plasma membrane .Although Dvl has been reported to associate with Frizzled receptors (Fz) during Fz-mediated Wnt signaling, it remains unresolved whether iLRP5-mediated β-catenin signaling is also Dvl-dependent. Previous findings have revealed the importance of Dvl in mediating LRP6-signalosome assembly Previous reports suggested LRP5 activation is regulated by phosphorylation of its cytoplasmic domain by membrane-associated CK1γ. 1408–1615) and iLRP5Δ1 (LRP51456–1615) containing two potential CK1 sites, iLRP5Δ2 (LRP51490–1615) containing one CK1 site, and iLRP5Δ3 (LRP51515–1615), iLRP5Δ4 (LRP51532–1615) and iLRP5Δ5 (LRP51553–1615) containing only “PPPSP” consensus sites without CK1 sites. Interestingly, we observed that the deletion of just one CK1 site in iLRP5 dramatically reduced β-catenin activation  in constructs iLRP5Δ4 and iLRP5Δ5, which do not carry any consensus CK1 phosphorylation sites It has been proposed that plasma membrane-associated CK1γ phosphorylates two Ser/Thr-containing motifs in LRP6 Fig. S4). Interestingly, both truncated LRP5-Δ2 and LRP5-Δ5 were constitutively phosphorylated even without dimerization, indicating that dimerization and CK1 phosphorylation rather than phosphorylation of PPP(S/T)P domains in LRP5 is required to induce iLRP5-mediated β-catenin activation.Subsequent co-immunoprecipitation studies between iLRP5 mutants and Axin revealed that the deletion of CK1 motifs and a domain (1516–1531) in iLRP5Δ4 and iLRP5Δ5, which contain at least three PPPSP motifs, also was correlative with downregulation of the Axin/iLRP5 interaction, suggesting that a possible defect of PPP(S/T)P phosphorylation in iLRP5 deletion mutants contributes to reducing Axin/iLRP5 interaction Fig. 5B.To further examine the possible correlation of iLRP5c or iLRP5c mutants with their subcellular localization, GFP fusion proteins with LRP5c deletion mutants were generated. Consistently, iLRP5Δ1 dimerization induced the distinct punctate cytoplasmic structures (similar to those observed with iLRP5c), whereas dimerization of iLRP5Δ2 and iLRP5Δ5 showed progressive loss of subcellular localization (iLRP5Δ3 showed the same pattern as iLRP5Δ2), indicating that re-localization to punctate structures after dimerization of iLRP5 exhibited a strong correlation with subsequent β-catenin activation Fig. 5C.1516MFYSSNIPATVRPYRPY1531) appears to possess important residues required for full iLRP5/Wnt signaling. Sequence homology analysis of this LRP5-derived region from various species as well as a comparison to highly homologous and functionally related co-receptor LRP6 supports a critical role for several residues . Interestingly, two highly conserved subregions within the sequence (YSS and YRPY) contain possible phosphorylation target sites, thus helping to target a limited mutagenesis study. Initially, we generated three membrane-targeted MF-hLRP5 variants, utilizing the lipid raft-targeting amino terminus of c-Fyn. In one, we replaced Y1518 with phenylalanine and adjacent serines with alanines . In a second, YRPY sequence was altered to FLPF  and the third clone possessed both FAA and FLPF mutant sequences. A TCF-reporter assay was used to measure constitutive canonical Wnt signaling of all 3 mutants relative to MF-hLRP5, containing the unmutated sequence . Curiously, although both FAA and FLPF mutants revealed reduced activity, the double-cluster mutant, containing FAA and FLPF residues had similarly reduced activity, possibly due to the inclusion of colocalized, endogenous LRP5 (or 6) in signaling-competent multimers.The previously unidentified region of LRP5  region for iLRP5 activity as well as for full activity of membrane-targeted MF-hLRP5, designed to approximate endogenous LRP5 function.To further dissect the importance of YSS and YRPY sequences, we generated three iLRP5 variants carrying the previously described mutations. We reasoned that non-membrane-localized iLRP5 variants would be less likely to interact with endogenous sequence. Interestingly, FAA, FLPF and FAA/FLPF double mutants now demonstrated distinct levels of Wnt activation upon cytosolic dimerization . Whereasb (MHC- class I) promoter . CID-mediated iLRP5 dimerization also resulted in cytoplasmic stabilization of β-catenin .In order to investigate the physiological relevance of the iLRP5 switch as a Wnt signaling activator, we designed transgenic mice, termed “Ubiquitous expression of inducible β-Catenin (Ubi-Cat)”. To affect broad somatic cell expression of iLRP5, we utilized the H-2Kpromoter . CID-medin vivo bioluminescent imaging. Ubi-Cat mice demonstrated broad iLRP5 expression via IVIS imaging . Moreover, iLRP5 expression via qRT-PCR was detected in multiple tissues of adult mice . Among the tissues examined, iLRP5 expression was highest in the pancreas, followed by testes, mammary gland, spleen and prostate.Next we examined transgene expression in Ubi-Cat mice. The Ubi-Cat construct is coupled to a click beetle red-shifted luciferase (CBR) reporter, permitting transgene expression detection via  imaging . Moreove−, Sca-1+, CD49f+  cells mixed in Matrigel, were transplanted into the backs of CD1nu/nu mice. The LSC grafts were analyzed 8 weeks after transplantation. In the WT control or Ubi-Cat-derived grafts in the absence of CID, glandular structures were generally composed of simple monolayer structures; however, hyperplasia was widespread in CID-treated grafts . iLRP5 activation in LSC grafts also resulted in enhanced cyclin D1 staining, suggesting that iLRP5 activation increases cyclin D1 activity . Analysis of p63 expression revealed that there was a two-fold increase in the number of p63+ cells in the iLRP5-induced, reconstituted grafts . Again, in vivo observations based on iLRP5 activation in LSCs support previously described results relying upon constitutively active β-catenin In order to examine the cell-autonomous effects of iLRP5 activation in LSCs during prostate regeneration, we used a prostate reconstitution assay. Multiple sub-Q grafts, containing 1×10d grafts . iLRP5 ad grafts . iLRP5 ain vivo prostate regeneration, we thought that this model could potentially be useful to examine the effect of Ubi-Cat-derived stroma in a developing prostate graft. Sca-1+, CD49f−, Lin− Ubi-Cat prostate stromal cells (1×105) were mixed with WT LSCs (1×105 cells) 1∶1 in Matrigel. The grafts were subsequently injected sub-Q into the backs of CD1nu/nu mice followed by 8 weeks of CID treatment. Visual inspection and weight measurement of the grafts indicated that the CID-treated samples were physically larger and heavier . Cross-sectional analysis of the grafts demonstrated that in the absence of CID or in control reconstituted grafts using WT stoma and Ubi-Cat LSCs, prostate acini were underdeveloped and lacking in prostate secretions. However, in CID-treated grafts, the stroma was more densely populated with cells and prostate ducts developed to a normal size (∼200 µm) that contained histologically normal prostate secretions . The observed phenotype suggests that iLRP5 is functional in Ubi-Cat stromal cells, as well as LSCs. Moreover, activation of Wnt signaling in prostate stroma via iLRP5 is sufficient to drive proper prostate development. These observations support an important role of Wnt signaling and its regulation during prostate development with possible cell autonomous functions in both basal LSCs and stromal cells.Prostate homeostasis is maintained via balanced communication between the stroma and the underlying epithelial compartment. Therefore, we were interested in the effects of iLRP5 induction in the prostate stroma. Since we have already established a working model for  heavier . Cross-sin vivo resulted in nuclear β-catenin localization by immunostaining . Activation of iLRP5 for up to 20 weeks further resulted in a modest hyperproliferation of prostate epithelial cells (Data not shown). Whereas age-matched, untreated mice showed slight proliferation among ventral lobe epithelial cells, treated mice reflected higher proliferative rates in both ventral and the dorsal lobes. The epithelial cells developed luminal hyperplasia and the normal single-cell layer organization of luminal cells was replaced by hyperplasia. We also observed a modest stromal thickening, consistent with reactive stroma. Furthermore, the severity of the proliferative phenotype was gradually enhanced with increased CID treatment, but not apparent in age-matched, untreated mice.To examine the outcome of iLRP5 activation in intact prostate tissue, a cohort of Ubi-Cat mice were treated bi-weekly with CID  for various durations. Animal cohorts were treated starting at 6 weeks of age for 4, 8, 20, 36, 44, and 52 weeks and prostate were analyzed upon sacrifice. Despite low-level, but prolonged β-catenin induction, treated mice appeared healthy during the course of these studies. CID-mediated iLRP5 activation . After 52 weeks of CID treatment, two of six mice had developed prostate adenocarcinoma, particularly in the ventral lobes. The ventral lobe was extremely disorganized and the basement membrane was severely disrupted or even absent in some acini . The dorsal and the anterior lobes were also affected. The epithelial cells had completely filled the lumens of the prostate ducts. In contrast, age-matched WT and untreated Ubi-Cat mice appeared relatively normal.Treatment of Ubi-Cat mice beyond 20 weeks resulted in an even more prominent hyperproliferative phenotype in the ventral and the dorsal lobes. Cells in the ventral lobe began to fill the lumen and coincided with significant stromal thickening, as well as a moderate level of acinar disorganization. Some nuclei were hyperchromatic and atypical, appearing elliptical by H&E analysis. Similarly, the dorsal lobes also demonstrated significant epithelial proliferation and stromal thickening, which was so severe in some foci that the stroma appeared to completely engulf the epithelial compartment. Moreover, the majority of acini were filled-in with epithelial and/or stroma cells By utilizing the prostate gland as a model for analysis of Wnt signaling induction in Ubi-Cat mice, we confirmed iLRP5 as a useful inducible β-catenin switch. CID-dependent changes in the prostatic lobes, from mild epithelial proliferation to adenocarcinoma, supported the physiological relevance of iLRP5-mediated Wnt signaling activation.3 transgenic cells on the left side and an equivalent number of WT cells on the right side. Transplanted mice were treated twice weekly with the AP20187 (CID) for 8-weeks prior to removal and analysis of grafted mammary cells. iLRP5-mediated Wnt pathway induction resulted in hyperplasia in 4 of 6 transplanted grafts, two of which developed mammary tumors . This was in comparison to normal mammary outgrowth observed in WT and untreated Ubi-Cat grafts. This mammary transplant data confirm that activation of canonical Wnt pathway signaling contributes to mammary gland hyperplasia and that Ubi-Cat transgenic mice can be used as a source of cells containing ligand-inducible ß-catenin expression.To better appraise the broader utility of the Ubi-CAT model, we choose to examine the outcome of another tissue known to be influenced by Wnt signaling. Maintenance of cellular homeostasis in mammary epithelial cells is highly dependent on modulation of Wnt signaling. Based on existing data, there appears to be a direct correlation with deregulation of Wnt signaling and breast cancer development Although it is widely accepted that the canonical Wnt/β-catenin signaling axis is initiated by Wnt-mediated Fz and LRP5/6 heterodimerization Drosophila melanogaster that includes Arrow (LRP5 homologue) and Dishevelled Our study also reveals novel plasma membrane-distal aspects of LRP5 function. It has been generally accepted that plasma membrane localization of LRP5 precedes phosphorylation by membrane-associated CK1α, which is required for Axin sequestration and β-catenin stabilization 1516MFYSSNIPATVRPYRPY1531), devoid of consensus CK1 phosphorylation sites, is essential for β-catenin induction, and intra-cytoplasmic localization. Nonetheless, we consistently observed that CK1 activity is critical for LRP5 function. Since dimerization of iLRP5 within intra-cytoplasmic compartments appears functionally intact, it is likely that cytoplasmic CK1 isoforms, associated with Dvl/Axin complexes, are able to induce LRP5 phosphorylation and activation, comparable to membrane-bound CK1. However, it remains to be tested what additional factor(s) target the newly identified signaling domain required for iLRP5 signaling and whether additional factor(s) are also critical for endogenous LRP5 function.Amino-terminal deletion and site-directed mutagenesis analysis of the cytoplasmic signaling domain of LRP5 revealed that a previously unidentified region of LRP5  and transforming growth factor β (TGF-β), for proper development. Multiple studies have described the presence of activated canonical Wnt signaling and its importance in prostate architecture organization and cell specification during prostate development and also androgen-dependent regeneration In this manuscript, we have provided two examples of Ubi-Cat-derived tissues that can be manipulated using a single dimerizing ligand. In addition to the effects of iLRP5 signaling in prostate epithelial and stromal tissue, we also investigated the outcome of iLRP5 induction within mammary cells. Mammary cell transplants into the mammary fat pad also indicated the cell autonomous effects of iLRP5 in mammary epithelial cells during development and homeostasis. In this case, induction of iLRP5 resulted in the formation mammary tumors, demonstrating that induction of unaltered β-catenin is sufficient to drive mammary tumor progression. This result further demonstrates the potency and efficacy of the iLRP5 switch as an inducer of the Wnt signaling pathway. Future studies will investigate the role of canonical Wnt signaling in other tissues in both reconstitution transplant models and in intact Ubi-Cat mice. Additional animal models based on tissue-specific regulatory elements or conditional, tissue-specific iLRP5 expression should further broaden the utility of this technology.Overall, this study has demonstrated the generation of a novel inducible Wnt signaling switch by manipulating the Wnt pathway co-receptor LRP5. We observed that membrane recruitment of iLRP5 via CID leads to activation of the Wnt signaling pathway. Oligomerization of LRP5 is also demonstrated to be a potent inducer of Wnt pathway induction, independent of membrane recruitment. This observation led us to generate an inducible Wnt switch (iLRP5) using only the cytoplasmic domain of LRP5. The activation of iLRP5 is achieved via CID technology, in which iLRP5-mediated Wnt signaling induction is dependent on exposure to synthetic homodimerizer drugs, like AP20187.in vitro and in vivo.Next, we investigated the molecular and biochemical events that follow upon iLRP5 activation, leading to Wnt signaling activation. We were able to demonstrate that iLRP5 interacts with Dvl; however, this interaction is not critical for the downstream activity of iLRP5. Instead, the iLRP5 switch has the ability to induce Wnt signaling by sequestering the Wnt inhibitory molecule, Axin. The iLRP5/Axin interaction appears sufficient to activate the Wnt pathway, even in the absences of full-length Dvl, suggesting that iLRP5 does not require Dvl activity or membrane localization. We also showed that iLRP5 phosphorylation via CK1 is required for proper iLRP5 activity. Finally, we generated the Ubi-Cat transgenic mouse model. In two distinct organ systems, Ubi-Cat mice demonstrated their utility as a practical tool for investigating the effect of Wnt signaling induction PP antibody was kindly provided by C Niehr All mice were housed at the Baylor College of Medicine (BCM) Transgenic Mouse Facility in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) for BCM. This study was IACUC reviewed and approved within protocol #AN-1428. HEK293  cells were maintained in DMEM, 10% FCS and antibiotics. Rapalog-B/AP22783 (Rap-B), FK1012 and AP20187 were provided by Ariad Pharmaceuticals . Rabbit anti-myc antibody (Covance/CRP), mouse anti-hemagglutinin (HA) epitope  and mouse anti-myc antibody  were used for immunoprecipitation and Western blotting. A phospho-specific anti-PPPSSalI-, and C-terminal NotI-linkered primers and subcloned into XhoI/NotI-digested pWG018-SEAP. Axin-myc and Dvl constructs were kindly provided by H Varmus (MSKCC). Full-length human LRP5 and LRP6 constructs, pCDNA6-hLRP5 and pCDNA6-hLRP6, were kindly provided by P McCrea (MDACC). The construction of Fyn-myristoylated FRBl2 (pBJ5/MF-FRBl2-E) was previously described F-LRP5c, the full-length cytoplasmic domain of LRP5c (LRP51408–16165) was PCR-amplified using N-terminal XhoI-, and C-terminal SalI-linkered primers and subcloned in-frame into XhoI-SalI digested pBJ5/MF-FRBl2-E.To generate TOP-SEAP and FOP-SEAP reporter plasmids, the LEF-responsive promoter (“TOP”) and mutant LEF-unresponsive promoter (“FOP”) from TOPflash or FOPflash  were PCR-amplified using N-terminal PK3-LRP5c (F3-LRP5c), pSH1-FPK3-GFP-LRP5c (F3-GFP-LRP5c) and pSH1-Fv′-Fvls-LRP5c (iLRP5), PCR-amplified LRP5c was subcloned into SalI-digested vectors pSH1-FPK3, pSH1-FPK3-GFP or pSH1-Fv′-Fvls. All truncated LRP5c mutants; iLRP5Δ1 (LRP51456–1616), iLRP5Δ2 (LRP51490–1616), iLRP5Δ3 (LRP51515–1616), iLRP5Δ4 (LRP51532–1616) and iLRP5Δ5 (LRP51553–1616) were PCR-amplified using primers containing N-terminal XhoI, and C-terminal SalI linkers and subcloned into the SalI site of pSH1-Fv′-Fvls-E or the SalI site 3′ of GFP in pSH1-FPK3-GFP-E To generate pSH1-FSEAP reporter assays in HEK293 cells were carried out in 6- or 12-well plates as described previously with minor modifications EcoRV-NotI (blunt) 2-kb fragment from H2K-I-LTR  was cloned into BamHI-digested (and blunted) KBPA vector to generate H2K-KBPA. Next, the 1.3-kb NotI-EcoRI digested iLRP5 fragment was removed from the pSH1-S-Fv′Fvls-LRP5-FLAGx2 construct. This fragment was cloned into NotI/MunI-digested pSH1(Mn)-IRES-Lip  to get pSH1/iLRP5-IRES. To obtain the reporter, we PCR-amplified the click beetle red-shifted luciferase (CBR) reporter using primers, 5CBRX: 5′-caagtcctcgagGTAAAGCGTGAGAAAAATGTCATC-3′ and 3CBRE: 5′-gacttgaattcTTACTAACCGCCGGCCTTCACC-3′ to get the 1664-bp CBR  fragment. The amplified product was digested via XhoI and EcoRI and the fragment was cloned into XhoI /EcoRI-digested pSH1/iLRP5-IRES to obtain pSH1/iLRP5-IRES-CBR. The NotI site was converted to the EcoRI site using primers Not2Eco5: 5′-ggccgcaagaattcaatc-3′ and Not2Eco3: 5′-ggccgattgaattcttgc-3′ to get pSH1(Nt)/iLRP5-IRES-CBR. This 3.6-kb fragment was then cloned into H2K-KBPA to get H2K/iLRP5-IRES-CBR. Transgene fragment was isolated by SpeI restriction digest. The fragment was gel purified using Qiagen gel purification kit . The purified fragments were used for injection into FVB strain-derived embryonic stem cells.The H2K-iLRP-I-CBR (Ubi-Cat) construct was generated in multiple cloning steps. First, an Ubi-Cat LSCs were cultured in Matrigel  as previously described in vivo reconstitution assays LSCs from 6–8 week-old adult WT or Ubi-Cat mice were mixed with adult prostate stroma or E-18 Rat UGSM. UGSM cells were prepared as described 5) and 1×105 UGSM or 1×105 WT LSCs and 1×105 adult prostate stroma were mixed and pelleted by centrifugation at 600 RCF. Pelleted cells were resuspended in 100 µl of Matrigel™ (BD Bioscience) on ice. The Matrigel:Cell mixture was injected subcutaneously into CD1nu/nu mice. Mice were treated with i.p. injection of AP20187  at 2 mg/kg in drug diluent  or drug diluent alone twice a week for 8 weeks. The grafts were isolated for further analysis.For 2O2 for 10 min. Sections were incubated with 1° antibody ON at 4°C. Primary antibody dilutions were as follows: β-catenin 1∶500 , p63 1∶500 , cyclin D1 (SP4) . Secondary antibody incubation was performed for 1 h at RT. Secondary antibodies (Invitrogen) were donkey anti-mouse Alexa Fluor 599, goat anti-rabbit Alexa Fluor 488. the slides were overlaid with ProLong® Gold anti-fade reagent with DAPI (Invitrogen).Formalin-fixed samples were embedded in paraffin and 5-µm sections were deparaffinized and hydrated in 100% and 95% EtOH steps and rinsed in water. Hydrated sections were stained with hematoxylin and eosin (H&E). Prior to IF and IHC, Sodium citrate (10 mM) antigen retrieval was performed, and sections were blocked with goat serum or M.O.M  kit for a minimum of 1 h at RT. When performing IHC, cellular peroxidases were neutralized using 3% HFor IHC, slides were incubated with secondary biotinylated goat anti-rabbit (1∶500 for 1 h at RT). Staining was performed by treatment with ABC reagent for 45 min followed by DAB for 3 min  and counterstained with hematoxylin.TCGCTGCAAACATTCCGGCCACTG CGAG-3′5′-GTACAACATGGACATGTTCT; iLRP5-YRPY→FLPF, TCTTGCCCTTCATCA TTCGA GGA ATGGCG-3′5′-CCGGCCACTGCGAGACCGT. (Bold characters denote mutated nucleotides.)The iLRP5 mutants were generated by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit . Oligonucleotides for different iLRP5 mutants were designed according to the manufacturer's protocol: iLRP5-YSS→FAA, 2, 1% Triton X-100). Cell lysates were crosslinked using 10 mM of disuccinimidyl glutarate  as suggested by the manufacturer. Samples were analyzed via Western Blot analysis.HEK293 cells were transfected with 1.5 µg of iLRP5 via FuGene 6 reagent (Roche) in 12-well format. 24-hr post-transfection, iLRP5 was activated via 100 nM/ml of AP20187 for an additional 24-hr. Prior to cell lysis on the following day, fresh AP20187 was added for an additional 2-hr. Cells were lysed in 50 µL of mild detergent  LiCl2-dependent, specific induction of TCF4/β-catenin-responsive reporter, TCF-SEAP, activity. 293 cells were transiently transfected with 1 µg of TOP-SEAP, FOP-SEAP reporter construct, or vector control. TOP-SEAP reporter activity was measured 24 hours after administration of indicated amounts of LiCl2. (B) Constitutive β-catenin induction by lipid raft targeting of the intact intracellular domains of LRP5 and LRP6 (MF-LRP5c and MF-LRP6c) but not by the triple PPPSP domain. 293 cells were transiently transfected with 1 µg of myristoylated EGFP (MF-EGFP), LRP5c (MF-LRP5c), LRP6c (MF-LRP6c), or the triple PPPSP domain of LRP5 (MF-LRP5-3xPPPSP) along with 1 µg of TOP-SEAP or FOP-SEAP reporter constructs. Twenty-four hours after transfection, SEAP reporter activity was measured. Data is representative of at least three independent experiments.(TIF)Click here for additional data file.Figure S2F-LRP5c-mediated β-catenin activation.Effect of Axin on M 293 cells were transiently transfected with 1 µg of TCF-SEAP reporter and 1 µg of MF-GFP (-) or MF-LRP5c expression vector along with indicated amounts (µg) of myc-tagged Axin. TCF-SEAP activity was measured 24-hours after transfection. The amount of protein expression was shown by anti-Myc and anti-HA blotting (bottom). Data is representative of at least three independent experiments.(TIF)Click here for additional data file.Figure S3Western blot analysis examining the expression of iLRP5/DIX, iLRP5/Axin and iLRP5/Dvl co-transfection experiments. 293 cells were transfected with 1 µg TCF-SEAP reporter along with 0.5 µg of iLRP5 and the indicated amounts (µg) of dominant negative disheveled-FLAG (A) or Axin-Myc constructs (B) or 0.5 µg disheveled-FLAG construct (C). Twenty-four hours after transfection, 100 nM CID was added to the cells and SEAP activity was measured following additional 24 h incubation. Cell lysate was obtained for Western blot analysis. Actin probe was used to assess the overall protein load in each lane.(TIF)Click here for additional data file.Figure S4A, B). Constitutive phosphorylation of PPPSP sites in iLRP5 mutants with or without dimerization. 293 cells were transfected with 1 µg of indicated mutants of iLRP5 along with 1 µg of the Axin expression construct. After 24-hour treatment with 100 nM AP20187 (+CID), whole cell lysates were immunoprecipitated with mouse anti-HA antibody  or rabbit anti-Myc antibody . Phosphorylation of PPPSP sites in iLRP5 mutants  and co-immunoprecipitation of LRP5 mutants with Axin  were analyzed by anti-pS1490  and anti-HA blot . Axin was immunoprecipitated from whole cell lysates with anti-Myc antibody (α-Myc). Co-immunoprecipitation of LRP5c was analyzed by anti-HA blot (α-HA). The amount of immunoprecipitated iLRP5 mutants  and iLRP5 mutant expression (B) was determined by HA blot .((TIF)Click here for additional data file.Figure S5iLRP5 promotes nuclear localization of β-catenin in prostate epithelial cells. Cellular localization of β-catenin in prostate tissue was analyzed via immunostaning. CID mediated iLRP5 dimerization results in activation of canonical Wnt singanling as suggested by presence of nuclear β-catenin (arrow heads).(TIF)Click here for additional data file.Figure S6iLRP5 promotes prostate hyperplasia. Activation of iLRP5 in Ubi-Cat mice for 36–44 weeks promotes proliferative phenotype within epithelial and stromal compartment. The cellular proliferation is more prominent in the ventral and dorsal lobes. Ventral and dorsal lobes demonstrate epithelial proliferation and significant stromal thickening. Bar = 200 µm.(TIF)Click here for additional data file.