During skeletogenesis, protein levels of β-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of β-catenin turnover by down-regulating intracellular levels of β-catenin.Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of β-catenin was used, our approach resulted in the accumulation of wild type β-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells.To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Our data indicate that a tight Apc-mediated control of β-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner. During vertebrate embryogenesis, the axial and appendicular skeleton develop through endochondral bone formation. In this process, mesenchymal cells aggregate to form a chondrocytic template that prefigures the shape of the future bone. At the periphery of this cartilaginous mold, osteoblasts differentiate to form the bone collar. The cartilaginous mold is eventually replaced by bone in a step-wise program. Besides chondrocytes and osteoblasts, the skeleton also contains osteoclasts, which are of haematopoietic origin and play pivotal roles in both cartilage and bone resorption and remodelling -3.Every step in the proliferation, differentiation, maturation, apoptosis, and resorption of both chondrocytes and osteoblasts is characterized by a specific transcriptional guideline . Sox9, aWnt14 overexpression in Collagen 2a1 (Col2a1)-expressing cells [Recently, based on mouse models, the canonical Wnt/β-catenin signaling pathway was found to act upstream of Sox9 and Runx2. In this pathway, in the absence of a Wnt signal, cytosolic β-catenin is degraded by the ubiquitination/proteasome system upon its phosphorylation at specific Ser-Thr residues by a destruction complex consisting of scaffold proteins such as Axin1, Axin2  and the adenomatous polyposis coli (APC) tumor suppressor, and two kinases, namely glycogen synthase kinase 3β (GSK3β) and casein-kinase 1α (CK1α). Binding of Wnt to a complex composed of the transmembrane frizzled receptor and low-density lipoprotein receptor-related protein 5 or 6 (LRP5 or 6) co-receptor results in inactivation of the destruction complex and accumulation of cytoplasmic β-catenin. Upon its nuclear translocation, β-catenin acts as transcriptional co-activator in complex with transcription factors of the TCF/LEF family, leading to transcriptional activation of Wnt target genes . In wildng cells .β-catenin in the limb and head mesenchyme before or during early mesenchymal condensations, Prx1-expressing and Dermo1-expressing skeletal precursors, respectively, differentiate into chondrocytes instead of osteoblasts [Osterix (Osx)-, Collagen 1a1 (Col1a1)- or Osteocalcin (Osc)-expressing osteoblasts suggest that Wnt/β-catenin signaling coordinates bone formation by controlling the differentiation and activity of both osteoblasts and osteoclasts in a sequential, stage-specific manner [It has been also demonstrated that β-catenin is required at an early stage to repress chondrocytic differentiation . Upon coeoblasts ,15. Finac manner ,17.Little is known about the mechanisms regulating β-catenin activity in skeletal precursors. Through its wide range of specific motifs and domains, APC is involved in multiple cellular processes such as signal transduction, cytoskeletal organization, apoptosis, cell adhesion and motility, cell fate determination, and chromosomal stability . HoweverAPC mutation frequently develop osteomas and dental anomalies [Apc1638N mutant mice occasionally develop osteomas . Homozygosity for the severely truncated ApcMin and for the more hypomorphic Apc1638N allele in the mouse results in a failure of primitive ectoderm development shortly after implantation, leading to lethality prior to gastrulation [ApcMin alleles, to more specific neuroectodermal, dorsal mesodermal, and endodermal defects  in case of two hypomorphic Apc1638N alleles [Familial adenomatous polyposis (FAP) patients heterozygous for an nomalies . Heterozrulation ,24. Muta alleles ,26. Oste alleles . HoweverApc, lose their differentiation capacity to both the chondrogenic and osteogenic lineage. Moreover, conditional Apc mutant ribs show enhanced osteoblast activity, while the mutant nasal septum displays chondrocyte dedifferentiation. These results provide the first genetic evidence that Apc plays a crucial role throughout mouse skeletogenesis by regulating the differentiation of skeletal progenitor cells and maintenance of chondrocytes.Here, we report that skeletal precursors of the axial and appendicular skeleton, when exposed to an uncontrolled rise of the β-catenin level due to conditional inactivation of Apc15lox allele where exon 15, encoding the majority of the coding region of Apc, and the polyadenylation signal, is flanked by loxP sites. Mice heterozygous and homozygous for the conditional Apc15lox allele did not show any major abnormalities or susceptibility to tumors. Δ15/+Apcmice, heterozygous for the Δ15Apcmutant allele obtained by germline Cre-mediated deletion of exon 15, developed multiple intestinal tumors at an early age similar toMin/+ Apcanimals. These results indicate that Cre-mediated recombination of the Apc15lox allele leads to inactivation of the Apc protein and to the constitutive activation of Wnt/β-catenin signaling .Recently, we (ECR-M and RF) generated a novel mouse model carrying a conditional Cre in transgenic Col2a1-Cre mice [LacZ reporter mice ("Rosaflox") [Cre;Rosaflox embryos expressed Cre specifically at all sites of endochondral bone formation  in mesenchymal condensations forming the sclerotome at E9.5  in cells of the osteogenic lineage prompted us to conclude that the Col2a1-Cre-mediated recombination occurred in skeletal precursors characterized by both a chondrogenic and osteogenic differentiation potential.Next, we investigated the temporal and spatial expression pattern of Cre mice  using Lasaflox") . Col2a1-ion Fig. . In acco somites , we dete9.5 Fig. . At E12.se lines ,30, we fApc15lox/15lox mice were bred with Col2a1-Cre mice to generate conditional heterozygous Col2a1-Cre;Apc15lox/+ mice. Microscopical analysis performed on Col2a1-Cre;Apc15lox/+ and control Apc15lox/+ embryos at various developmental stages  displayed a normal spatio-temporal expression of all chondrogenic and osteogenic markers investigated (data not shown).Cre;Apc15lox/+ mice  and 11 Apc15lox/+ mice  were monitored for 12 weeks after birth. Mice of both genotypes were healthy, similar in appearance, size, body length/weight ratio and growth rate (data not shown). We next assessed bone architecture in these animals by micro-computed tomography (μCT) of the distal femora. No difference was detected between Col2a1-Cre;Apc15lox/+ mice and gender-matched Apc15lox/+ control littermates with respect to bone mineral density, trabecular bone volume fraction, trabecular number, trabecular thickness, and trabecular separation  and 5 Apc15lox/+ male mice were followed for 24 months. At the end of this period, animals were sacrificed and tissues were analyzed microscopically using hematoxiline/eosine-stained sections. No important abnormalities could be distinguished in the skull, ribs, vertebral column and long bones. We evenly detected in both groups signs of cartilage degradation, fibrosis, and osteochondritis, pathological findings which most likely were all age-related (data not shown). Altogether, we considered conditional heterozygous Apc mutant embryos as controls for the next experiments.To study postnatal growth and bone acquisition, 18 Col2a1-Cre;Apc15lox/+ mice were crossed with Apc15lox/15lox mice to generate conditional homozygous Col2a1-Cre;Apc15lox/15lox mice (1:4). None of these mice were found at one month of age among 77 liveborn offspring. Of 27 dead pups found within the first month after delivery, only 5 pups on the day after delivery were Col2a1-Cre;Apc15lox/15lox. To further investigate the Col2a1-Cre;Apc15lox/15lox phenotype, embryonic litters at various developmental stages were isolated. Eight of 31 embryos isolated between E16.5 and E19.5 were Col2a1-Cre;Apc15lox/15lox (26%). We concluded that conditional homozygosity for this Apc mutant allele was perinatally lethal. At E12.5, Col2a1-Cre;Apc15lox/15lox embryos, although normal in size, displayed poor mandible and limb outgrowth compared to control littermates  and Alizarin red  of embryos at E14.5 revealed a clear difference in size between Col2a1-tes Fig. . All muttes Fig. . Distorttes Fig. , while n6.5 Fig. . At this6.5 Fig. . Mutant red Fig. . In the red Fig. .Cre;Apc15lox/15lox embryos, we analyzed vertebra formation at E12.5 and E14.5, and humerus development at E16.5 using IHC for β-catenin in combination with Alcian blue staining, and in situ hybridization (ISH) for several chondrocyte- and osteoblast-specific genes. Strongly elevated levels of β-catenin were seen at all sites of endochondral ossification in the Col2a1-Cre;Apc15lox/15lox embryos at E12.5, E14.5 and E16.5, indicating efficient Col2a1-Cre-mediated Apc inactivation.Lack of functional Apc results in accumulation of cytoplasmic β-catenin, which subsequently translocates into the nucleus. This process can be well detected by immunohistochemistry (IHC). To investigate endochondral bone formation in Col2a1-Sox9 and Col2a1. In marked contrast, mutant sclerotomal cells failed to condense into skeletal primordias. They showed strong nuclear β-catenin staining and displayed a mesenchymal-like spindle shape morphology. These cells expressed neither Sox9, nor Col2a1, implying that conditional loss of functional Apc in skeletal precursors inhibited mesenchymal cell condensation and chondrogenic differentiation. Next, we investigated whether these cells had switched their commitment to the osteogenic lineage due to the accumulation of β-catenin. Surprisingly, they did not express the early osteoblast markers Runx2 and Col1a1, suggesting that β-catenin accumulation due to Apc inactivation impaired osteogenic differentiation of skeletal precursors as well  and mature chondrocyte markers, like Indian hedgehog (Ihh) and Collagen 10a1 (Col10a1), indicating a normal progression of endochondral ossification   Fig. . In contp9) Fig. . Similarp9) Fig. . In addited Fig. , suggest4.5 Fig. .Apc inactivation on differentiation of skeletal precursors in long bones and vertebrae, proximal ribs of Col2a1-Cre;Apc15lox/15lox embryos at E16.5 showed clearly enhanced mineralization upon skeletal staining , indicating that, unlike in the long bones and vertebrae, Apc inactivation in skeletal precursors of the proximal ribs did not impair osteoblastogenesis  at similar levels compared to control cartilage (data not shown). The abundant presence of a bone matrix combined with evidence of functional osteoclasts suggested that the β-catenin-positive osteoblasts were sclerotic.Despite the inhibitory effect of ing Fig. . Therefoing Fig. . Mutant ing Fig. , 7A. β-Cing Fig. . At E16.ing Fig.  and 7E–Gely Fig. . Interessis Fig. . Since ISox9 and Col2a1 .The presence of β-catenin-positive cells in the chondrogenic islands suggested that these cells, due to mosaicism of Apc in murine Col2a1-expressing cells. Our data indicate that the Col2a1 promoter is suitable for this study, since Cre-mediated recombination starts very early (E9.5) in skeletal precursor cells that have not yet committed to the chondrogenic or the osteogenic lineage, consistent with previous findings in other Col2a1-Cre lines [According to most of the transgenic mouse studies reported, levels of β-catenin, the effector of the canonical Wnt ligands, need to be downregulated in skeletal precursor cells to enable chondrogenic differentiation, whereas elevated β-catenin levels promote differentiation into osteoblasts ,15,35,36re lines ,30.Apc does not result in a detectable level of its target β-catenin as determined by IHC. Moreover, heterozygous Col2a1-Cre-mediated Apc inactivation does not interfere with embryonic skeletal development, postnatal growth or bone acquisition up to 24 months of age, as determined by histological and μCT analysis. Our data imply that the level of Apc protein produced by a single functional Apc allele is sufficient to mediate appropriate β-catenin degradation. This is in agreement with normal body weight, size, and growth of young ApcMin/+ mice [Conditional heterozygous inactivation of n/+ mice .Apc results in a strongly elevated level of (wild type) β-catenin in skeletal precursors, leading to greatly impaired embryogenesis and perinatal lethality. The significantly reduced size and the vast range of skeletal malformations in these embryos is most likely due to the specific Col2a1-Cre activity in skeletal primordias at a very early embryonic stage starting at E9.5 resulting in massive β-catenin accumulation in the developing endochondral skeleton. Probably several factors, like the open rib cage and the severe malformation, from E14.5 on have led to the perinatal lethality. The loss of the multiple β-catenin-independent functions of the Apc protein might have contributed to the gravity and complexity of the skeletal phenotype observed in Col2a1-Cre;Apc15lox/15lox mice as well [Col2a1 expression is not completely restricted to skeletal tissues during mouse embryogenesis [Apc in other Col2a1-Cre-expressing cell types.In marked contrast, conditional inactivation of  as well . Moreoveogenesis , we can β-catenin leads to decreased chondrocyte differentiation in Prx1-expressing and Col2a1-expressing cells [Prx1-expressing cells, suggesting that activation of β-catenin negatively affects skeletogenesis [β-catenin in Prx1-expressing cells leads to increased expression of not only chondrocyte but also early osteoblast markers [Wnt/β-catenin signaling represents a mechanism in mesenchymal precursor cells for selecting between chondrocytic and osteoblastic fates. This key regulating role in lineage commitment has been attributed to β-catenin. Indeed, conditional gain-of-function mutation of ng cells ,15. Howeogenesis ,15. In a markers .Apc mutations to differentiate into bone and cartilage [We report here that in the vast majority of endochondral skeletal elements, precursor cells lacking functional Apc express strong nuclear β-catenin staining and fail to differentiate into both chondrogenic and osteogenic lineages. These data are in line with the inability of mouse embryonic stem cells carrying specific bi-allelic artilage . Our datartilage . This coApc in skeletal precursors, we find a different phenotype in the proximal ribs. Notwithstanding the cartilaginous structure at E14.5, proximal ribs of Col2a1-Cre;Apc15lox/15lox mutants at E16.5 show abundant bone matrix deposited by osteoblasts, invariably expressing high levels of nuclear β-catenin. Since osteoblasts do not express Col2a1, these cells are most likely derived from Col2a1-expressing skeletal precursors lacking functional Apc. This implies that, in contrast to other skeletal elements, skeletal precursors of the proximal ribs are able to escape from the noxious effects of strongly elevated β-catenin levels on differentiation of precursor cells by an as yet unknown mechanism. Since Ihh expression is normal in the non-recombined neighbouring chondrocytes, we speculate that Ihh may be a prime target for inducing osteoblastogenesis in the recombined precursor cells counteracting the noxious effect of β-catenin.Although in the vast majority of the endochondral skeleton both chondrogenic and osteogenic differentiation is inhibited due to loss of functional Apc or constitutively active β-catenin in already differentiated osteoblasts, resulting in dramatically increased bone deposition [Despite the evidence of functional osteoclasts, the intensely ossified proximal ribs show a strongly diminished bone marrow cavity, rendering it likely that the increased bone formation is due to osteosclerosis. These observations are in agreement with other data, showing that enhanced canonical Wnt signaling can increase bone mass through stimulation of osteoblast activity rather than inhibition of osteoclast formation and activity -41. Suchposition ,42.We have found clear evidence for the occurrence of chondrocyte dedifferentiation due to β-catenin accumulation in the nasal septum. Morphologically characterized chondrocytes, which were nuclear β-catenin-positive, lacked expression of typical chondrocyte markers. Furthermore, they were imbedded in an ECM containing significantly less proteoglycans.Apc after completion of the initial stages of chondrocyte differentiation. Mouse models with an increased level of β-catenin in Col2a1-expressing cells show accelerated chondrocyte maturation [Apc does not result in chondrocyte maturation but in chondrocyte dedifferentiation. Our data suggest that accumulated β-catenin triggers this dedifferentiation program not only through inhibition of chondrogenic marker expression but also by enhancing the loss of ECM presumably through stimulation of matrix-degrading enzymes. It has been demonstrated that β-catenin increases expression and activity of a number of enzymes involved in matrix degradation [Cre;Apc15lox/15lox embryos, however, the presence of these cells was most pronounced in the nasal septum. Altogether, our data indicate that Apc is required to suppress β-catenin for maintenance of the chondrocytic phenotype.Given the noxious effect of increased β-catenin levels on chondrocyte formation , theturation ,12. We hradation -45. β-Caradation . DediffeApc gene results in a heterogeneous skeletal phenotype. Based on our results, we postulate that Apc-mediated control of the dosage of transcriptionally active β-catenin protein is directive for the differentiation program of skeletal precursor cells. In the vast majority of the skeletal precursors, loss of functional Apc leads to a strongly increased β-catenin level, resulting in the formation of an undifferentiated mesenchymal cell, which lacks differentiation potential for both osteogenic and chondrogenic lineages. When the inhibitory effect of a strongly increased β-catenin level in the skeletal precursors is reduced, highly active osteoblasts arise. Strong repression of β-catenin in these precursors is required for chondrogenesis. Support for our hypothesis on the importance of the dosage of Apc and β-catenin is provided by observations in Col2a1-Wnt14 transgenic mice [Wnt14 expression resulting in a high level of β-catenin block differentiation of skeletal precursors into chondrocytes or osteoblasts, whereas lower levels of Wnt14 expression result in enhanced ossification. We provide evidence that Apc plays a crucial role in modulating the β-catenin level during mouse skeletogenesis in a spatio-temporal regulated manner. In skeletal precursor cells, Apc is required for differentiation into both chondrocytes and osteoblasts. In addition, Apc is essential in chondrocytes to maintain their phenotype and enable their maturation.We show here for the first time that Apc, by negatively controlling the levels of β-catenin, is a critical regulator of the differentiation of skeletal progenitor cells. Conditional inactivation of the mouse nic mice . Higher Apc15lox/+ mouse was generated by gene targeting in IB10 embryonic stem cells, using a 22.5 kb targeting vector containing loxP sites flanking the last exon of Apc, i.e. exon 15. LoxP sites were inserted in the BglII site of intron 14 and in the ApaI site approximately 350 bp downstream of the Apc polyadenylation signal. Exon 15 of the Apc gene encodes for codons 660 to 2842 of the Apc protein and harbours all the functional domains of Apc involved in β-catenin regulation as well as the C-terminal domains binding to microtubules, DLG, and EB1. Therefore, following Cre-mediated deletion of exon 15, functionality of the remaining protein will be fully impaired with respect to the main function of Apc, i.e. β-catenin regulating. Moreover, as deletion of exon 15 also removes the polyadenylation signal, no stable mRNA is produced and as a consequence no stable truncated protein will be generated. A full description of this new conditional Apc mouse model is currently in preparation . Col2a1-Cre mice [Apc15lox/15lox mice. Of the offspring, Col2a1-Cre+/-;Apc15lox/+ mice were mated with Apc15lox/15lox mice to obtain Col2a1-Cre+/-;Apc15lox/15lox mice. LacZ reporter mice were obtained from Dr. Xiaohong Mao [All animal studies were approved by the ethical committee of the Leiden University Medical Centre and complied with national laws relating to the conduct of animal experiments. The Cre mice  were matSkeletons of mouse embryos were stained with Alcian blue and Alizarin red for cartilaginous and mineralized tissues, respectively, according to standard procedures . For mic2O2 in 40% methanol/60% TBS for 30 minutes to reduce endogenous peroxidase activity. For antigen retrieval the sections were boiled in Tris-EDTA pH 9.0 for 20 minutes. Blocking was performed with 5% blocking buffer for 30 minutes at 37°C (Boehringer Ingelheim). Sections were incubated with the primary mouse monoclonal antibody against β-catenin  overnight at 4°C, followed by incubation with the second antibody biotin-conjugated rabbit anti-mouse IgG  for 45 minutes at 37°C. The biotinylated proteins were detected by incubation with horseradish peroxidase-conjugated streptavidin  for 30 minutes at 37°C and visualized with DAB (Sigma). After counterstaining with Alcian blue for 15 minutes and hematoxylin for 1 minute, sections were dehydrated and embedded in Histomount (BDH). For in situ hybridization, digoxigenin-labeled single-stranded RNA probes were prepared using a DIG RNA labeling kit (Boehringer) following the manufacturers' instructions. All probes are available upon request. In situ hybridization was carried out as described [Whole mount β-galactosidase staining was performed as described , from E1escribed . Images Cre mice were provided by TK and HMK; mutant mice were generated and genotyped by CAJB and ECR-M; embryo experimental work and analysis were performed by RLM; μCT analysis was performed by GR; data interpretation was carried out by RLM assisted by MK, GH, MAV, PA, CWGML, RF, JMW, and ECR-M; the manuscript was written by RLM with the assistance of all co-authors. All authors read and approved the final manuscript.The studies were designed and initiated by MK as principal investigator, with the help of RF and ECR-M; Col2a1-


Understanding how the mechanical microenvironment influences cell fate, and more importantly, by what molecular mechanisms, will enhance not only the knowledge of mesenchymal stem cell biology but also the field of regenerative medicine. Mechanical stimuli, specifically loading induced oscillatory fluid flow, plays a vital role in promoting healthy bone development, homeostasis and morphology. Recent studies suggest that such loading induced fluid flow has the potential to regulate osteogenic differentiation via the upregulation of multiple osteogenic genes; however, the molecular mechanisms involved in the transduction of a physical signal into altered cell fate have yet to be determined.Using immuno-staining, western blot analysis and luciferase assays, we demonstrate the oscillatory fluid flow regulates β-catenin nuclear translocation and gene transcription. Additionally, real time RT-PCR analysis suggests that flow induces Wnt5a and Ror2 upregulation, both of which are essential for activating the small GTPase, RhoA, upon flow exposure. Furthermore, although β-catenin phosphorylation is not altered by flow, its association with N-cadherin is, indicating that flow-induced β-catenin signaling is initiated by adherens junction signaling.We propose that the mechanical microenvironment of bone has the potential to regulate osteogenic differentiation by initiating multiple key molecular pathways that are essential for such lineage commitment. Specifically, non-canonical Wnt5a signaling involving Ror2 and RhoA as well as N-cadherin mediated β-catenin signaling are necessary for mechanically induced osteogenic differentiation. Primarily, loss of function mutations of the canonical receptor, LRP5, result in osteopenia or familial cases of osteoporosis, while gain of function mutations result in an autosomal high bone density trait Although canonical Wnt signaling via β-catenin translocation may promote differentiation in specific conditions, it should be noted that β-catenin translocation to the nucleus may also be regulated by cadherin signaling ex vivo mineralization The non-canonical Wnt signaling pathway, which does not involve β-catenin, is transduced through Frizzled and Ror1 or Ror2 co-receptor complexes and has the potential to initiate the RhoA, JNK and calcium signaling pathways among others In this study, we examine the potential of mechanical stimuli to regulate classically defined canonical and non-canonical signaling pathways in C3H10T1/2 murine MSCs by investigating the induction of β-catenin translocation and expression of Wnt associated proteins with fluid flow. Additionally, we determine if β-catenin and Wnt5a signaling are involved in mechanically induced osteogenic differentiation via Runx2 upregulation. Furthermore, we investigate Wnt5a's potential to activate RhoA, a possible non-canonical effector shown to be necessary for flow induced Runx2 expression, and determine if the tyrosine receptor-type kinase, Ror2, is involved. Finally, we examine the molecular mechanism governing β-catenin signaling and translocation to the nucleus. We propose that non-canonical Wnt5a signaling involving Ror2 and RhoA as well as N-cadherin mediated β-catenin signaling are necessary for mechanically induced osteogenic differentiation.2.C3H10T1/2 mesenchymal stem cells (ATCC) were cultured in Eagle's Basal medium (BME) with 2 mM L-glutamine (Sigma). Media was adjusted to contain 2.2 g/L sodium bicarbonate and supplemented with 10% fetal bovine serum (Hyclone), 1% penicillin and streptomycin (Invitrogen). Cells were maintained at 37°C and 5% CO5′- AAU CUU CCA CYY CAC CUG C- 3′) or 0.20 µM scrambled control siRNA  were transfected into cells using 10 µl of XtremeGene (Roche Molecular Systems) and OptiMEM (Invitrogen) media. After 4 hours of incubation, media was put on cells and flow experiments were performed 48 hours later. Neither the scrambled siRNA, Wnt5a siRNA, nor Ror2 siRNA had any significant effect on overall cellular morphology. To inhibit β-catenin signaling, cells were incubated with 15 µg/ml of endostatin (Sigma) for 24 hours prior to flow.To prevent Wnt5a or Ror2 signaling, 0.20 µM siRNA targeting Wnt5a , 0.20 µM siRNA for Ror2 . The dynamic flow profile was sinusoidal at a frequency of 1 Hz. For β-catenin studies, cells were exposed to flow for 35 minutes and lysed immediately. For Wnt expression studies, cells were exposed to 15, 30 or 45 minutes of flow and lysed immediately. For RhoA assays, cells were exposed to 1 hour of flow and lysed immediately. For Runx2 expression, cells were exposed to 1 hour of flow followed by a 30 minute incubation in fresh BME growth media, then lysed.Cells were subcultured on fibronectin coated glass slides (76×35×1 mm) at 140,000 cells/slide. Fluid flow was applied to cells that were 80–90% confluent at the time of experimentation. A previously described fluid flow device was used to deliver oscillatory fluid flow to C3H10T1/2 progenitor cells 5′-AGA AGG CAC AGA CAG AAG CTT GA- 3′; Runx2 Reverse 5′- AGG AAT GCG CCC TAA ATC ACT-3′; Wnt5a Forward 5′- CTC CTT CGC CCA GGT TGT TAT AG -3′; Wnt5a Reverse 5′- TGT CTT CGC ACC TTC TCC AAT G -3′; Wnt3a Forward 5′- ATA GCC TGC ATC CGC TCT GA -3′; Wnt3a Reverse 5′- TGG TGA CCA TTG CCT CAA CA -3′; Ror2 Forward 5′-TGG AAC TGT GTG ACG TAC CC -3′; Ror2 Reverse 5′-GCG AGG CCA TCA GCT G -3′. Each sample was analyzed in triplicate. Gene expression levels were normalized against 18S rRNA assessed from the same sample tube.Cells were lysed after flow exposure and total RNA was isolated using Tri-Reagent (Sigma). The 260/280 absorbance ratio was measured for verification of the purity and concentration of the RNA. Reverse transcription was completed using GeneAMP RNA PCR Core kit (Applied Biosystems) with 0.75 µg of RNA. Analysis by quantitative real-time RT-PCR  was conducted using Taqman PCR Master Mix and a 20× 18S primer and probe (Applied Biosystems), or by using SYBER green PCR master mix with primers and probes developed by Operon technologies for Runx2, Wnt3a, Wnt5a, Ror2. The primer sequences were: Runx2 Forward To detect β-catenin translocation, cells were exposed to oscillatory fluid flow for 35 minutes, then immediately fixed in 3.7% formaldehyde and membranes were removed with 0.1% Triton-X in PBS. Cells were incubated with 1∶100 monoclonal mouse anti-β-catenin antibody  and 1∶100 donkey IgG in primary blocking solution  overnight. Cells were then washed with 1× PBS for 10 minutes for a total of 3 times and incubated with FITC-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Labs) for 30 minutes. Cells were washed 3 times for 5 minutes in PBS. Cover slips were mounted on the slides using 35 µl of Vectashield mounting medium (Vector Laboratories) and sealed with nail polish.2, 10 µg/ml aprotinin/leupeptin, and 1 mM PMSF (Sigma)) at 4°C. Samples were centrifuged for 3 minutes at 3000 rcf at 4°C. 30 µl of supernatant was removed and used to determine total RhoA; the remaining volume of supernatant was incubated with rhotekin binding domain-beads (Millipore) for 45 min at 4°C. After incubation, samples were centrifuged for 3 minutes at 3000 rcf, and washed three times using IP buffer ), and suspended in SDS-PAGE buffer. RhoA was assayed by Western blot.RhoA-GTP was measured by pull-down assay  were immediately trypsinized and centrifuged at 5000 rcf for 5 minutes and supernatant was removed. Nuclear and cytoplasmic protein were isolated using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology). The kit protocol was followed with the addition of proteinase inhibitor, PMSF and sodium orthovate to the reagents: CER I and NER.2, 0.1 mM sodium orthovanadate, 10 µg/ml aprotinin/leupeptin, and 1 mM PMSF (Sigma)) and resuspended in SDS page buffer.To determine the effects of oscillatory fluid flow on N-cadherin and β-catenin association, cells were lysed immediately following the cessation of flow (1 slide per sample) using 100 µl of lysis buffer. Samples were sonicated for 5 seconds and centrifuged at 3000 rcf for 3 minutes. The supernatant was incubated with 25 µl Protein G sepharose beads (Amersham Pharmacia) for 15 min and centrifuged for 2 min at 14,000 rcf. The supernatant was incubated with 2.5 µl of anti-β-catenin antibody for 30 min, and then 50 µl of Protein G sepharose beads was added and incubated for 1 hour. The beads were washed four times with IP buffer  were lysed immediately after 35 minutes of flow in 100 µl of lysis buffer, sonicated and centrifuged for 3 minutes at 3000 rcf. Supernatants were used for protein quantification.Protein concentration in the supernatant was determined using a Bradford protein assay kit . Suspended samples were electrophoresed through NuPAGE 4–12% Bis-Tris polyacrylamide gels (Invitrogen) and were transferred electrophoretically onto nitrocellulose membranes in blocking buffer (Pierce). After washing, membranes were incubated with primary antibody overnight at 4°C under gentle rocking. Incubation with a HRP-conjugated anti-goat/mouse IgG (1∶2000 dilution) was carried out for 1 hour at room temperature under gentle rocking. Signals were visualized using a chemiluminescent ECL substrate . Densitometric analysis was performed using ImageQuant software. Protein levels were normalized using total RhoA (for RhoA assays), beta actin (for β-catenin assays) or β-catenin (for β-catenin phosphorylation and N cadherin co-immunoprecipitation).Antibodies used were: β-catenin , N-cadherin , RhoA , Serine 33 Phosphorylated β-catenin .Luciferase detection was determined with the Dual Light System (Applied Biosystems). Mouse L cells transfected with TOPflash (described by Mikels and Nusse Data are expressed as mean±standard error. A student T-test was used to compare control cells and cells exposed to fluid flow. A p<0.05 was considered significant.Immuno-staining of control cells and cells that had been exposed to 35 minutes of oscillatory fluid flow indicated that, with mechanical stimulation, there is a greater amount of β-catenin localized to the nucleus . To valiBased on our finding that β-catenin signaling is induced by oscillatory fluid flow, we next wanted to determine if oscillatory fluid flow regulated the expression profile of several known Wnt proteins associated with osteogenic differentiation or stem cell renewal that may initiate β-catenin nuclear translocation . Cells wTo determine the role of Wnt5a and β-catenin signaling in flow induced osteogenic lineage commitment, cells were treated with several inhibitory factors, exposed to fluid flow, and Runx2 expression was compared to that of control cells. Primarily, we interrupted Wnt5a signaling using siRNA technology that resulted in a significant 55% decrease in Wnt5a mRNA expression levels. Inhibiting Wnt5a signaling via siRNA significantly reduced the cell's ability to upregulate Runx2 in the presence of mechanical stimulation. Specifically, scrambled siRNA treated cells exposed to fluid flow had a 2.2±0.2-fold increase in Runx2 expression compared to control cells. However, Wnt5a siRNA treated cells had no difference in Runx2 expression between flow-exposed and control cells . To inveGiven that Wnt5a and β-catenin signaling are necessary for flow-induced osteogenic differentiation, and that Wnt5a has the potential to induce both the canonical and non-canonical pathways, we next investigated whether flow-induced Wnt5 signaling was activating the canonical or non-canonical pathways . To deteGiven that β-catenin signaling was not the target of Wnt5a, we next determined if Wnt5a signaling was necessary for flow induced RhoA activation. Furthermore, we also used Ror2 siRNA, which significantly decreased Ror2 mRNA expression, to determine if this receptor was, in fact, necessary. For these studies cells treated with scrambled, Wnt5a, and Ror2 siRNA were exposed to oscillatory fluid flow and a pull down assay utilizing agarose beads with the rhotekin binding domain enabling the isolation of active RhoA was used to isolate GTP-bound RhoA. Analysis of the blots indicates that the 1.7±0.1-fold increase in active RhoA observed in scrambled treated cells was significantly greater than Wnt5a and Ror2 siRNA treated cells . Both ofTo determine the mechanism inducing β-catenin signaling, we examined both β-catenin phosphorylation with flow, mediated by canonical Wnts, and N-cadherin/β-catenin association, mediated by cadherin signaling . WesternThe mechanical microenvironment of bone has the potential to regulate osteogenic lineage commitment by initiating multiple key molecular pathways that are essential for cell fate commitment. Understanding what molecular mechanisms are involved in lineage commitment will not only enhance the current understanding of stem cell biology but also the field of regenerative medicine. In this study, we show that mechanical stimulation is a potent signal that initiates both non-canonical Wnt signaling and β-catenin signaling. Additionally, flow induced β-catenin signaling initiates the transcription of β-catenin/TCF/LEF downstream genes, including Runx2. Furthermore, flow upregulates Wnt5a and Ror2, both of which are necessary for flow induced RhoA activation and ultimately, Runx2 expression Our study is the first to suggest that Wnt5a is upregulated by mechanical stimuli and is necessary for flow induced osteogenic lineage commitment. These results are consistent with previous reports that have demonstrated Wnt5a is important in MSC osteogenic differentiation Oscillatory fluid flow induces β-catenin nuclear translocation and the initiation of TCF/LEF/β-catenin transcription, which is necessary for flow induced Runx2 expression. However, oscillatory flow does not appear to regulate canonical Wnt3a, and other Wnt proteins associated with osteogenesis were not expressed in C3H10T/2 MSCs. Furthermore, β-catenin phosphorylation is not altered upon flow exposure suggesting that flow-induced β-catenin signaling is not due to canonical Wnt signaling. Interestingly, our results show that the association between N-cadherin and β-catenin is decreased upon mechanical stimulation. This suggests the possibility that flow-induced liberation of β-catenin in adherens junctions or cell-cell contacts may be the first step in enabling β-catenin to accumulate in the cytoplasm and ultimately translocate to the nucleus to initiate transcription in response to mechanical stimulation. Our findings support previous work conducted by Norvell and colleagues demonstrating that upon mechanical stimulation with unidirectional fluid flow, β-catenin is also induced to translocate to the nucleus and initiate transcription of Cox2, another osteogenic gene, in MC3T3-E1 pre-osteoblast cells in vitro and in the intact limb bud in vivoAdditionally, we show that both β-catenin signaling, which may be mediated by cadherin signaling, and Wnt5a/RhoA signaling is necessary for flow induced osteogenic differentiation. This is indicative that multiple intracellular pathways must be initiated for lineage commitment to occur. In fact, studies indicate that stabilized β-catenin has the potential to induce Runx2 and Osteopontin expression, but it does not induce Osteocalcin expression In summary, we determined that the mechanical microenvironment that MSCs are exposed to upon migration into bone may initiate multiple intracellular signaling pathways required for osteogenic lineage commitment. Specifically, flow weakens N-cadherin association with β-catenin enabling β-catenin to translocate to the nucleus and initiate gene transcription, which is necessary for flow induced Runx2 expression. Furthermore, oscillatory fluid flow additionally up-regulates Wnt5a and Ror2, both of which are necessary for flow induced RhoA activation. This flow-induced Wnt5a signaling and RhoA activation are necessary for osteogenic differentiation due to flow


For microbial invaders to establish an infection, they must overcome the defenses mounted by one of the body's staunchest defenders, the T cell. T cells come in several different flavors, each with a specific role to play. Cytotoxic T cells battle intracellular pathogens, while helper T cells (Th cells) direct antibody production and orchestrate the overall immune response by secreting intercellular messaging proteins known as cytokines. Th cells can differentiate further into two major types (known as Th1 or Th2 cells), which can be distinguished from one another based on which cytokines they secrete.PLoS Biology, Dimple Notani, Sanjeev Galande, and colleagues provide novel insights into how the transcription factor Special AT-rich Binding Protein 1 (SATB1), known to be required for T cell development, affects the outcome of β-catenin signaling at the transcription level. They show how β-catenin and SATB1 interact in T cells to affect Th cell growth and differentiation.In order to mount an effective immune response, T cells of all kinds must be able to recognize foreign invaders while also ignoring body-derived materials. Therefore, before they differentiate into cytotoxic T cells or Th cells, immature T cells (known as thymocytes because they are found in the thymus) go through a series of checkpoints to ensure they fulfill these requirements. During this lengthy checking process, thymocytes require external signals to help support their growth. One of these support signals is provided by Wingless (Wnt) proteins. First characterized in flies as a mutation that impairs thoracic segment development, Wnt signaling works similarly in all organisms; when soluble Wnt proteins bind to their receptors on cells, they initiate a signaling cascade that ultimately results in the accumulation of the protein β-catenin in the cells' nucleus. There, β-catenin binds to and activates other proteins (known as transcription factors) that control the expression of genes needed for cell growth. In this issue of SATB1 is expressed primarily in thymocytes and in Th2 cells. It is known to bind to DNA, tethering DNA to the protein scaffolding that lines the nucleus. This helps organize DNA into tidy loops for better control of gene expression. SATB1 also serves as a molecular adaptor for several other proteins that work to pack DNA into an inactive state. In this way, SATB1 is thought to repress the activity of genes found nearby its DNA binding sites. However, some of the genes SATB1 represses are known to be upregulated by Wnt signals – a fact that led Notani and colleagues to explore whether (and how) SATB1 and Wnt signaling might interact in T cells.When the authors examined the nuclei of Wnt-stimulated thymocytes under a microscope, they found that SATB1 and β-catenin  are present in the same parts of the nucleus, suggesting these two proteins might directly interact. Subsequent experiments showed that SATB1 and β-catenin are not just in proximity to each other, but actually bind directly to one another: the first half of SATB1 protein binds to β-catenin, while the last part of β-catenin binds to SATB1.The finding that SATB1 and β-catenin bind to one another led the researchers to examine whether Wnt signaling might cause SATB1 to recruit β-catenin to genes. They found that Wnt signaling causes an increase in SATB1 DNA binding by promoting the deacetylation of SATB1—a modification that is known to increase SATB1's affinity for DNA. What's more, this increased SATB1 binding to genes is mirrored by increased levels of β-catenin on those same genes. Collectively, these data suggest that Wnt signaling increases SATB1 binding to DNA, and that SATB1 then recruits β-catenin to DNA. Once β-catenin is bound to SATB1, β-catenin can recruit additional partners to help stimulate gene expression, thereby indirectly converting SATB1 from a repressor to an activator of gene expression.The authors' findings add an important new detail to our understanding of the role played by both SATB1 and Wnt in thymocytes. But, because SATB1 is specifically expressed in Th2 cells, Notani and colleagues also wanted to explore what contributions SATB1  might make to Th2 cell differentiation. They found that Th cells of all kinds make Wnt proteins and so can stimulate themselves with Wnt signals. However, Th2 cells experience higher levels of Wnt signaling than do Th1 cells. Therefore, in Th2 cells, SATB1 and β-catenin can cooperate to promote the expression of GATA3, another transcription factor that is known to control the differentiation and development of Th2 cells, thereby promoting the production of Th2-characteristic cytokines. These new insights will allow researchers to better understand how T cell development is controlled by chromatin organizing factors in collaboration with various signaling processes, how this process can go awry, and the diseases that can result from T cell–related immune disorders (including autoimmunity or immune deficiencies).H2 Differentiation in Wnt-Dependent Manner. doi:10.1371/journal.pbio.1000296Notani D, Gottimukkala KP, Jayani RS, Limaye AS, Damle MV, et al. (2010) Global Regulator SATB1 Recruits β-Catenin and Regulates T


In serrated adenoma (SA), a newly recognised subtype of colorectal adenoma, APC mutations are uncommon, and the contribution of β-catenin to tumorigenesis remains unclear. We compared intracellular localisation of β-catenin and presence of mutations in exon 3 of β-catenin between 45 SAs, with 71 conventional adenomas (CADs), and eight carcinomas invading the submucosa (SCAs). Widespread or focal nuclear β-catenin expression was demonstrated in 7% of SAs (three out of 45), 61% of CADs (43 out of 71), and 88% of SCAs (seven out of eight). Cytoplasmic immunostaining for β-catenin was demonstrated in 16% of SAs (seven out of 45), 77% of CADs (55 out of 71), and 88% of SCAs (seven out of eight). No mutation in exon 3 of β-catenin was found in SAs or SCAs, while 7% of CADs (five out of 71) had β-catenin mutations. No nuclear or cytoplasmic expression of β-catenin was observed in the hyperplastic or conventionally adenomatous epithelium of mixed-type SAs. These findings suggest that β-catenin mutation is unlikely to contribute to the tumorigenesis in SA, and that intracellular localisation of β-catenin may not be associated with an early event of the tumour progression in most SAs.Intracellular redistribution of  APC) suppressor gene is an initial event in the development of most colorectal tumours  family, and activation of this transcriptional complex resulting in constitutive transcriptional activation of downstream target genes that regulate cell proliferation or apoptosis . As phosphorylation of these sites is apparently necessary for APC-induced degradation of β-catenin , such as Ki-utations . Given autations , the difAPC gene mutation is uncommon in SA. However, the contribution of β-catenin to tumorigenesis in SA remains unclear. The aim of this study was to examine intracellular localisation of β-catenin and to identify any mutations in exon 3 of β-catenin in SAs, by comparing the findings with those in CADs and carcinomas invading the submucosa (SCAs).Recently, n=44) or transanal resection (n=1) at Showa University Hospital between September 1997 and December 2001 were analysed in this study. CADs (n=71) and SCAs (n=8) obtained endoscopically or surgically were studied for comparison. Informed consent was obtained from all patients. We excluded patients who had familial adenomatous polyposis, hereditary nonpolyposis colorectal cancer (HNPCC), or hyperplastic polyposis  were cut from the paraffin block to be prepared for haematoxylin and eosin (H&E) staining and immunostaining. All H&E-stained sections were reviewed by a senior pathologist (TY), who was blinded to colonoscopic findings. The histologic diagnosis of SA was based on the definition of Figure 1Specimens resected or obtained by endoscopy were fixed in 10% buffered formalin for 24 h, dehydrated, and embedded in paraffin. Serial sections (3 −1 sodium citrate buffer (pH 6.0) for 15 min to retrieve antigens. Endogenous peroxidase activity was inhibited by incubation with 0.3% hydrogen peroxidase in methanol for 5 min. Sections were incubated with anti-β-catenin antibody  for 90 min. Sections were then incubated with horseradish peroxidase (HRP)-binding amino-acid polymer for 30 min . Colour was developed by staining with diaminobenzidine solution. Sections were lightly counterstained with haematoxylin. Phosphate-buffered saline (pH 7.2) was used for rinsing between each step.Deparaffinised, rehydrated sections were heated in a microwave oven in 0.01 mol lEach immunostained section was examined under light microscopy and evaluated by a senior pathologist (TY) according to the modified scoring method of Figure 2μm thick) were obtained from an archival block of formalin-fixed, paraffin-embedded tumour tissue for each polyp type. One section was stained with H&E, and the percentage of tumour cells was estimated by microscopic examination. Representative tumour samples contained a minimum of 80% tumour cells. After deparaffinisation, DNA samples were extracted from the remaining four sections using standard proteinase K–phenol–chloroform methods.To extract genomic DNA, five sections  using laser-capture microdissection. Deparaffinised sections were stained with H&E, followed by three dehydration steps of 60 s each in 70, 95, and 99.5% ethanol, with final dehydration in xylene. Once air-dried, the stained tissues were laser-capture microdissected by a PixCell II LCM system following the manufacturer's protocols . After the tumour cells had been collected into a 0.5-ml reaction tube, DNA was isolated by adding 50 β-catenin was examined for mutations using fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. Primers used for PCR were 5′-TGATTTGATGGAGTTGGACA-3′ (forward) and 5′-CTGTTCCCA-CTCATACAGGA-3′ (reverse). The PCR reaction was carried out in a 20 μl final volume, containing 50 ng DNA, 0.5 U Takara Ex Taq , 1 μmol of each primer, 2.5 mM of each dNTP, and 10 × Ex Taq buffer (Takara). The following cycling protocol was used: 94°C for 1 min, followed by 40 cycles of 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min, and then a final extension at 10°C for 60 min. PCR products were visualised and purified by electrophoresis on 2% agarose gels for 40 min at 100 V, and stained with ethidium bromide. These PCR products were used to detect β-catenin mutation by fluorescence-based SSCP analysis using an automated DNA sequencer , with an external cooling bath. Peak patterns were analysed using the ALFwin Fragment Analyser program (Amersham Pharmacia Biotech). Shifted peaks indicated the mutations in the DNA fragments. Nucleotide sequences of DNA fragments with shifted peaks were determined as described previously , in contrast to 61% of CADs (43 out of 71) (P<0.0001). All but one SCA specimen was positive for nuclear staining. In SAs and CADs, nuclear expression of β-catenin was observed more frequently in large (>10 mm) than in small (<10 mm) lesions. However, these size-related differences were not statistically significant.Table 2β-catenin in SAs, CADs, and SCAs. Cytoplasmic accumulation of β-catenin was demonstrated in 16% of SAs (seven out of 45) and 77% of CADs (55 out of 71: P<0.0001). All but one SCA specimen was positive for cytoplasmic staining. In CADs, cytoplasmic immunostaining for β-catenin was detected more frequently in large (⩾10 mm) than in small (<10 mm) CADs (P=0.0029). However, no significant difference was noted in cytoplasmic immunostaining of β-catenin between large and small SAs. No significant difference in cell-membrane expression of β-catenin could be found among SAs, CADs, and SCAs.Table 3β-catenin was found in SAs or SCAs, while 7% of CADs (five out of 71) had β-catenin mutations. Considering the size of CADs, β-catenin mutations were detected in 8% of small CADs  and 6% of large CADs . Table 4β phosphorylation region.No mutation in exon 3 of β-catenin was observed in the hyperplastic or conventionally adenomatous epithelium in the 20 mixed polyps. In the carcinomatous epithelium of mixed-type SAs, nuclear β-catenin-positive cells were focally detected, and cytoplasmic staining for β-catenin was observed . Abnormal distribution of β-catenin was detected more frequently in large CADs (⩾10 mm) than in smaller CADs. However, nuclear overexpression of β-catenin generally was not observed in our SAs; even focal nuclear staining was found in only 7% (three out of 45). Cytoplasmic accumulation of β-catenin was observed in 16% of SAs (seven out of 45). The nuclear and cytoplasmic localisation of β-catenin in SA thus differs from observations in CADs and SCAs.Recent reports have described nuclear expression of β-catenin in 7% of sporadic CADs (five out of 71). As in previous studies . This disagreement with the previous study  (β-catenin immunostaining in sporadic MSI-H cancers (β-catenin mutation is found in sporadic MSI-H cancers, the subjects are young and probably have HNPCC (β-catenin mutation is not found in 27 samples of microsatellite stable CRC. It can therefore be inferred that β-catenin mutation is implicated in only a very small proportion of CRC that may be a subset of HNPCC cases.β-catenin in colorectal tumour cells usually results from mutations in the APC or β-catenin genes (β-catenin protein has been shown to activate its target gene expression by the complex formation with a family of nuclear transcription factors. APC mutations are present in about 80% of sporadic colorectal adenomas and cancers, while in recent studies (APC mutations have been rare in SAs. We observed no mutation of β-catenin in SAs. Nuclear or cytoplasmic expression of β-catenin was significantly less common in SAs than in CADs or SCAs. Furthermore, no nuclear or cytoplasmic immunostaining of β-catenin was observed in adjacent hyperplastic or conventionally adenomatous glands in mixed-type SAs. Thus, the signalling pathway involving APC and β-catenin seems unlikely to be important for tumorigenesis in most SAs.Recent experimental and clinical analyses have indicated that cytoplasmic expression and nuclear translocation of  studies , APC mutβ-catenin mutation occurs in a small subset of CAD, and CAD initiated by β-catenin mutation may rarely progress to CRC. From the preceding observation, it can be deduced that β-catenin is not implicated in the initiation of SAs, or that SAs rarely progress to carcinoma. We demonstrated that no mutation of β-catenin was detected in SAs. Furthermore, when we separately examined β-catenin mutation in serrated and nonserrated areas of mixed-type SAs, no β-catenin mutation was found in either area. Thus, mutations in exon 3 of β-catenin are unlikely to contribute to tumorigenesis in SA. In fact, it has been previously shown that β-catenin and APC mutations are uncommon in SAs (The previous study (β-catenin was demonstrated in the carcinomatous component of this lesion, cytoplasmic accumulation of β-catenin was observed. Neither nuclear nor cytoplasmic staining was found in the adjacent serrated epithelium. The immunohistologic heterogeneity demonstrated in this tumour implies that intracellular redistribution of β-catenin might be a late event in the development of colorectal cancer. However, only one such case of carcinoma associated with SA was analysed in this study, so further investigation of these genetic changes is required to clarify the mechanism of tumorigenesis in SA.We found one mixed-type SA with two histologically distinct areas representing carcinoma and SA. Although focally nuclear expression of β-catenin in the development of SA using immunohistochemical and genetic methods. Intracellular localisation of β-catenin in SAs differed from that in CADs or SCAs, and no β-catenin mutation was observed in SAs. We concluded that β-catenin mutation is unlikely to contribute to tumorigenesis in SA. Further, intracellular localisation of β-catenin may not be associated with an early tumorigenic event in most SAs.In summary, we evaluated the status of


Homeodomain-interacting protein kinase-2 (HIPK2) plays an essential role in restraining tumor progression as it may regulate, by itself or within multiprotein complexes, many proteins (mainly transcription factors) involved in cell growth and apoptosis. This study takes advantage of the recent finding that HIPK2 may repress the β-catenin transcription activity. Thus, we investigated whether HIPK2 overexpression may down-regulate vascular endothelial growth factor (VEGF) levels (a β-catenin target gene) and the role of β-catenin in this regulation, in order to consider HIPK2 as a tool for novel anti-tumoral therapeutical approaches.The regulation of VEGF expression by HIPK2 was evaluated by using luciferase assay with VEGF reporter construct, after overexpression of the β-catenin transcription factor. Relative quantification of VEGF and β-catenin mRNAs were assessed by reverse-transcriptase-PCR (RT-PCR) analyses, following HIPK2 overexpression, while β-catenin protein levels were evaluated by western immunoblotting.HIPK2 overexpression in tumor cells downregulated VEGF mRNA levels and VEGF promoter activity. The VEGF downregulation was partly depending on HIPK2-mediated β-catenin regulation. Thus, HIPK2 could induce β-catenin protein degradation that was prevented by cell treatment with proteasome inhibitor MG132. The β-catenin degradation was dependent on HIPK2 catalytic activity and independent of p53 and glycogen synthase kinase 3β (GSK-3β) activities.These results suggest that VEGF might be a target of HIPK2, at least in part, through regulation of β-catenin activity. These findings support the function of HIPK2 as tumor suppressor and hypothesise a role for HIPK2 as antiangiogenic tool in tumor therapy approaches. Homeodomain interacting protein-kinase 2 (HIPK2) has been initially identified as corepressor for various homeodomain-containing transcriptional regulators . In the c-myc, cyclin D1, uPAR, c-jun, and fra-1 [One essential element of the Wingless-Wnt signalling pathway is β-catenin, a potent oncogene whose accumulation has been implicated in tumorigenesis in a wide variety of human cancers . β-catennd fra-1 -17, invoin vivo and in vitro [Among the β-catenin target genes is vascular endothelial growth factor (VEGF) , a potenin vitro . Tumor pin vitro . The inhin vitro . These fThese findings, along with a recent study showing that HIPK2 represses the transcription of the β-catenin target cyclin D1 , prompte2 at 37°C. Proteasome inhibitors MG132  was prepared as 50 mM stock in DMSO, stored at -20°C and diluted into the medium at 2.5 μM for 6 h. The treatment with 30 mM LiCl  was for 16 h.Human lung adenocarcinoma H1299 and human breast cancer MCF7 cell lines were cultured in RPMI-1640 , human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium , all supplemented with 10% heat-inactivated fetal bovine serum (GIBCO-BRL) plus glutamine and antibiotics in humidified atmosphere with 5% COTransient transfection was carried out using the N,N-bis-(2-hydroxyethyl)-2-amino-ethanesulphonic acid-buffered saline (BBS) version of the calcium phosphate procedure . The amoCells were harvested in TRIzol Reagent (Invitrogen) and total RNA was isolated following the manufacturer's instructions. The first strand cDNA was synthesized according to the manufacturer's instructions . Semi-quantitative RT-PCR was carried out by using HOT-MASTER Taq (Eppendorf) with 2 μl cDNA reaction and genes specific oligonucleotides under conditions of linear amplification. The sequence of the primers used for RT-PCR was as follow: human β-catenin forward: 5'-GAAAATCCAGCGTGGACAATGGCTACT-3' and reverse 5'-ACC-ATAACTGCAGCCTTATTAACC-3'; human VEGF forward: 5'-CCTGGTGGACATCTT-CCAGGAGTA-3'; and reverse: 5'-TCACCGCCTCGGCTTGTC-ACA-3'. The VEGF amplification leads to doublets representing the 165 and 121 VEGF isoforms, as previously shown . DNA proTotal cell extracts were prepared by incubating at 4°C for 30 min in lysis buffer  plus a mix of protease inhibitors . Proteins were then separated by SDS-PAGE and blotted onto nitrocellulose (Bio-Rad). The membranes were probed with a primary antibody followed by a secondary antibody conjugated with horseradish peroxidase. The antibodies used were: rat monoclonal anti-HA (Roche Diagnostics); rabbit polyclonal anti-cyclin D1 ; mouse monoclonal anti-tubulin ; rabbit polyclonal anti-β-catenin (Santa Cruz Biotechnology), rabbit polyclonal anti-phospho-GSK3α/β , and mouse monoclonal anti-GSK3β (Santa Cruz Biotechnology). Immunoreactivity was detected with the ECL chemoluminescence reaction kit . Data presented are representative of at least three independent experiments.t test. Data are expressed as mean ± SD. A value of p ≤ 0.05 was considered statistically significant.Continuous variables were analyzed by the Student High levels of VEGF expression have been associated with tumor angiogenesis and therefore with tumor progression in a wide variety of human cancers . To deteThese results indicate that HIPK2 overexpression decreased both VEGF mRNA and β-catenin protein levels and β-catenin transcription activity. The possible link between HIPK2 and β-catenin-mediated transcriptional regulation of VEGF was next evaluated.The effect of HIPK2 on β-catenin-mediated transcription activation was first evaluated on artificial TCF/LEF-1 luciferase reporter plasmid. To this end, H1299 cells were co-transfected with TOPFlash reporter and a combination of HIPK2 and β-catenin expression vectors. As shown in Figure Altogether, these data show that HIPK2 inhibited β-catenin-mediated VEGF transcription and that HIPK2 catalytic activity was likely involved in this regulation.Western blot analysis was performed in order to test whether the inhibition of HIPK2-dependent β-catenin activity could depend by β-catenin protein destabilization, as HIPK2 can regulate protein turnover through phosphorylation-targeted/proteasomal degradation ,6. To thSince HIPK2 is a potent activator of p53 function  and it hThese data suggest that HIPK2 down-regulated β-catenin levels through proteasomal degradation system, an effect dependent on HIPK2 catalytic activity and independent of p53 and GSK3β activities.The results presented in this study revealed a novel target of HIPK2 oncosuppressor function that is VEGF, and strengthened the role of HIPK2 as transcriptional repressor of β-catenin-function. We showed that HIPK2 down-regulated both endogenous VEGF mRNA levels and the VEGF levels induced by β-catenin overexpression, in different tumor cells, highlighting interplay between HIPK2-mediated β-catenin regulation and VEGF expression. Thus, we have found that HIPK2 might induce β-catenin proteasomal degradation and inhibit its transcription activity.The tumor progression is often dictated by increased vascularity following VEGF up-regulation . Thus, dIncreasing evidence indicates that HIPK2 is able to modulate the transcription activity of a growing number of transcription factors involved in cell growth and apoptosis [reviewed in ref. , thus itCollectively, the data presented in this study indicated that HIPK2 could repress the β-catenin transcription activity and downregulate VEGF expression in tumor cells. Constitutive activity of β-catenin can exert both proliferative and antiapoptotic effects  as well The authors declare that they have no competing interests.RP carried out cell transfection, immunoblotting analysis, and luciferase assays, LN contributed to cell transfection, cell treatments, RT-PCR, and densitometric analyses. GDO supervised experimental work and wrote the manuscript. All authors read and approved the final manuscript.


A recent study has shown that phosphorylated c-Jun (p-c-Jun) interacts with TCF4 to form a complex that cooperatively enhances their transcriptional activity in the presence of β-Catenin, and that their interaction is critical for mouse intestinal tumorigenesis. To determine the significance of these three proteins in human colorectal tumors, we analyzed their nuclear expression by immunohistochemistry.we analyzed their nuclear expression by immunohistochemistry using paraffin-embedded specimens of 68 resected colorectal tumors, which consisted of 19 adenomas, 14 high-grade intraepithelial neoplasia (HGINs) and 35 adenocarcinomas. We also analyzed the expression of MMP7, which has functional AP-1 and TCF binding sites in its promoter.Expression of p-c-Jun, TCF4 and β-Catenin were significantly higher in adenomas than in the adjacent normal epithelia. Expression of p-c-Jun and β-Catenin in HGINs and adenocarcinomas were also significantly higher than in the adjacent normal epithelia. p-c-Jun expression, but not TCF4 and β-Catenin, was higher in adenomas and HGINs than in adenocarcinomas, in which p-c-Jun expression was negatively correlated with pT stage progression. Furthermore, significant correlations of expression were observed between p-c-Jun and TCF4 , TCF4 and β-Catenin , p-c-Jun and MMP7 , and TCF4 and MMP7 , respectively.These results suggest that nuclear expression of p-c-Jun, TCF4 and β-Catenin have important roles in human colorectal tumor development and that p-c-Jun may play a pivotal role in the earlier stages of tumor development. Apc) tumor suppressor gene, which results in deregulation of the APC/β-Catenin/T-cell factor 4 (TCF-4) signaling pathway ) ,27,32. Sp < 0.05 was considered statistically significant. Spearman's rank correlation test was used to determine the correlation with staining intensity and percentage score.Wilcoxon matched pairs signed ranks test was used to determine the differences between the tumors and the adjacent normal epithelia. The Mann-Whitney U test and Kruskal-Wallis test were used to determine the difference between two groups and among more than two groups, respectively. For the immunofluorescent staining of p-c-Jun, TCF4 and β-Catenin, the sections were subjected to antigen retrieval by heating in 0.01 M sodium citrate (pH 6.0) in a microwave oven for 25 min. The slides were allowed to cool for an additional 20 min in citrate buffer. The sections were permeabilized in PBS containing 0.2% Triton X-100 (PBT) for 20 min at room temperature. Nonspecific binding sites were blocked with normal goat serum for 30 min at room temperature. The sections were incubated at 4°C overnight with a combination of the primary rabbit polyclonal antibody against p-c-Jun (1:2 dilution) and one of the mouse monoclonal antibodies against TCF4 (1:4 dilution) and β-Catenin (1:60 dilution). After washing, the sections were incubated for 90 min at room temperature with the mixture of Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody and Alexa Fluor 568-conjugated goat anti-mouse IgG antibody which were highly cross-absorbed . The sections were then evaluated and photographed under a fluorescence microscope. To determine specificity of the immunofluorescent staining, serial sections were similarly processed except that primary antibodies were omitted.In the tumor cells of adenomas, HGINs and adenocarcinomas, staining for p-c-Jun was mainly detected in the nucleus, while staining was observed in the nucleus and cytoplasm for TCF4, and in the nucleus, cytoplasm and cellular membrane for β-Catenin. Staining for MMP7 was observed in the cytoplasm of the tumor cells. Representative immunostaining patterns for these proteins are shown in Fig. p-c-Jun, TCF4 and β-Catenin in the adenomas, and p-c-Jun and β-Catenin in the HGINs and adenocarcinomas showed significantly higher scores than in their adjacent normal colorectal epithelia, respectively (p < 0.05 by Wilcoxon matched pairs signed ranks test)  to evaluate results, high correlations were observed for each proteins between staining intensity and percentage score in adjacent normal epithelia and tumors of all three types  Fig. . ExpressTo investigate the relationships of p-c-Jun, TCF4, and β-Catenin with MMP7, whose promoter contains both functional AP-1 and TCF binding sites , we analTo determine whether p-c-Jun, TCF4 and β-Catenin are colocalized in the nuclei, several specimens of adenocarcinoma that expressed each protein were examined by immunofluorescence. A representative specimen demonstrated that p-c-Jun and TCF4 were colocalized predominantly in the nuclei in a majority of the tumor cells. Although β-Catenin was predominantly located in the cellular membrane of the tumor cells, it was also colocalized with p-c-Jun in some parts of the nuclei  scores.Click here for fileSupplemental Table S2: Correlation between staining intensity and percentage of positive cells (percentage score) in all colorectal tumors and adjacent normal epithelia .Click here for fileSupplemental Table S3: Nuclear expression of p-c-Jun, TCF4, β-Catenin and MMP7 in tumor compared with adjacent normal colorectal epithelia by using percentage scores.Click here for fileSupplemental Figure S1: Nuclear expression of p-c-Jun, TCF4 and β-Catenin in adenomas (n = 19), HGINs (n = 14) and adenocarcinomas (n = 35) by using percentage of positive cells (percentage score). Only p-c-Jun expression was significantly different. Adenomas and HGINs showed significantly higher percentage scores than adenocarcinomas (p = 0.01). Horizontal lines, median; boxes, 25% to 75% range; brackets, 10% to 90% range; circles, points outside the 10% to 90% range. *p < 0.05 by Kruskal-Wallis test.Click here for fileSupplemental Figure S2: Relationship between nuclear expression of p-c-Jun, TCF4 and β-Catenin and the pT and pN stages in adenocarcinomas (n = 35) by using percentage score. a) p-c-Jun expression was significantly negatively correlated with the pT stage progression . b) No significant correlation was observed between p-c-Jun, TCF4 and β-Catenin expression, and the pN stage . Horizontal lines, median; boxes, 25% to 75% range; brackets, 10% to 90% range; circles, points outside the 10% to 90% range. *p < 0.05 by Mann-Whitney U test.Click here for file


CTNNB1) and secreted frizzled-related protein-1 (sFRP1), in human trabecular meshwork (TM) cells. In addition, the effect of oxidative stress on Wnt signaling was evaluated.The aim of this study was to examine the expression of genes related to the Wnt signaling pathway, such as β-catenin (2O2 for 30 min. Actin staining was carried out to verify cell response to oxidative stress. Western blotting was used to measure Wnt-related protein levels after H2O2 treatment.All experiments were conducted using second- or third-passaged human TM cells. cDNA was prepared from total RNA extracted from cells by means of reverse transcription. PCR was then performed to determine the presence of Wnt genes. For oxidative stress, TM cells were treated with 1 mM of HPositive PCR products were detected for a total of 25 Wnt and Wnt-related genes in human TM cells. Most of the genes identified belonged to the Wnt/β-catenin pathway. Members of the β-catenin-independent noncanonical pathways were also found. Oxidative stress did not result in significant changes in β-catenin and sFRP1 protein levels.Genes related to canonical and noncanonical Wnt pathways are expressed in human TM cells. It appears that all three Wnt pathways are operative in the TM system. Oxidative stress, while thought to play a role in the development of glaucoma, had little effect on the Wnt activity in TM cells. The trabecular meshwork (TM) located at the chamber angle of the eye is the major site for regulation of the aqueous humor outflow . Cells iThe Wnt gene family encodes secreted glycoproteins that are highly conserved across a variety of species . These p2+ leads to release of intracellular Ca2+ and involves activation of protein kinase C and Ca2+/calmodulin-dependent protein kinase II. The Wnt/Ca2+ pathway has implications on cell proliferation and cell movement [The Wnt genes signal through a β-catenin-dependent canonical pathway as well as two β-catenin-independent noncanonical pathways. Central to the canonical pathway is the regulation of β-catenin activity. In the absence of Wnt ligands, β-catenin accumulated in the cytoplasm is phosphorylated by glycogen synthase kinase3β (GSK3β) in a multiprotein destruction complex composed of proteins, including axin and the adenomatous polyposis coli (APC) tumor suppressor protein, and is thereby marked for degradation through the ubiquitin–proteasome system. When the Wnt ligand binds to membrane receptor frizzled (Fzd) and the low-density lipoprotein receptor-related protein 5/6 (LRP5/6), the cytoplasmic target of Fzd, disheveled (Dsh), is activated to suppress the GSK3β activity. As a result, β-catenin is neither phosphorylated nor degraded, and the excess β-catenin is translocated into the nucleus [movement .Many extracellular inhibitors of Wnt signaling have been reported. One example is the secreted frizzled-related protein (sFRP), which antagonizes Wnt signaling by sequestering the Wnt protein in the extracellular matrix . AnotherA possible role of Wnt signaling in the outflow system has been suggested recently . An incrTM tissues were dissected from corneo-scleral rims obtained from donors 32, 33, 43, 51, and 58 years of age . The tissues were plated onto Falcon Primaria flasks . At least 2 ml of complete medium ,21 conta2O2; Sigma) in serum-free complete medium (Sigma) for 30 min [To study the effects of oxidative stress, cells at confluence for at least 2 days were treated with 1 mM hydrogen peroxide  in a volume of 50 μl. The primers, designed using the OligoPerfect™ Designer (Invitrogen) program, were purchased from either Sigma or IDT . The sequences of primer pairs for target genes along with the annealing temperatures and expected product sizes are listed in Total RNA was isolated from cultured TM cells, using the RNeasy mini kit . The RNA concentration was measured using an ND-1000 spectrophotometer . At least 1 μg of RNA was used for cDNA synthesis, employing random hexamers and the Superscript RT first strand synthesis kit (Invitrogen). PCR was conducted on aliquots of cDNA samples by using Multigene thermocycler . Each reaction mixture contained 5–10 μM of gene-specific primer pairs, 0.5–1 μg of cDNA, and PCR master mix containing 1.5 mM MgClTM 100-bp ladder markers  were resolved on 1% agarose gels. The gel images were captured with the Gel Doc 2000 image analyzer . Sequencing analyses were performed to verify the identities of all PCR products. At least two independent experiments, using cells from two different donors, were performed.Thermocycling conditions were 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, primer-specific annealing temperature for 30 s, 72 °C for 30 s, and a cycle of 72 °C for 10 min. Negative controls with RNA samples that were not subjected to reverse transcription were included for all PCR experiments. PCR products, along with Marker VI  or GeneRulerWnt2, Wnt2b, Wnt3, Wnt3a, Wnt4, Wnt10b, Wnt11, sFRP1, sFRP2, β-catenin (CTNNB1), and LRP5 [TCF1 gene expression as this transcription factor has been shown to be expressed in the kidney [A human breast adenocarcinoma cell line, MCF-7, is known to express and LRP5 -29. MCF-e kidney .2O2 treatment, using the CelLytic M reagent (Sigma) in the presence of protease inhibitors (Roche). Total protein was quantified by the bicinchoninic acid assay . At least 10 µl of cell lysate was used to determine the protein content. Bovine serum albumin was used as a standard. Protein concentration was determined using Tecan GeniosPro microplate reader . Equal amount of protein from each sample resolved on a 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel under reducing conditions was electroblotted onto a Protran nitrocellulose membrane . The membrane was blocked with 5% nonfat dry milk and incubated with monoclonal anti-β-catenin  or polyclonal anti-sFRP1 . Horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG  was used as the secondary antibody. Protein bands were detected using HyGLO Chemiluminescent HRP antibody detection kit . The blot was also stripped using ImmunoPure IgG Elution buffer (Thermo Scientific) for 30 min at room temperature and reprobed with polyclonal anti-glyceraldehyde 3-phosphate dehydrogenase  for protein loading control. Densitometry was performed and Wnt protein levels were normalized against that of GAPDH. At least three independent experiments were conducted. Statistical analyses were performed using the Student’s t test.Lysate was prepared from TM cells after H2O2, TM cells in chamber slides were fixed immediately after (0 h) or 4 h later with fixative containing 2% paraformaldehyde, 0.08 M lysine, and 10 mM sodium periodate [After a 30-min treatment with 1 mM Heriodate  for 20 mCTNNB1, GSK3β, APC, four TCF transcription factors, and five inhibitors was examined by PCR analyses. All PCR products were subjected to gel electrophoresis. Positive products of expected sizes for 25 Wnt and Wnt-related genes were detected . The reduction persisted for at least 4 h (4-h time point).Actin staining was conducted to verify cell response to oxidative stress . A reducIn the present study we examined the expression of Wnt components in human TM cells. Wnt signaling is documented to play an important role in various biologic processes, including differentiation, development, regulation, and apoptosis of cells -33.Wnt2b, Wnt3, Wnt5a, and Wnt5b), three Dsh genes , seven members of the receptor complex , CTNNB1, GSK3β, APC, three transcription factors , and five inhibitors  are expressed (Four Wnt ligands (xpressed  in humanWnt5a, Wnt5b, Dsh3, Fzd2, and Fzd4, have been shown to be members of the noncanonical Wnt/PCP and/or Wnt/Ca2+pathways. Still another few have been linked to more than one pathway (2+ pathway and acts as an antagonist in the Wnt/β-catenin pathway in 3T3-L1 preadipocytes [Among the 25 genes we identified, most are members of the canonical β-catenin-dependent pathway . Several pathway . One exaipocytes . In addiWnt2b, Wnt5a, Fzd1, Fzd2, Fzd7, and sFRP1 is consistent with that reported previously by Wang and co-workers [Dkk2 and Fzd7 were additionally found in a gene microarray study by Zhao et al. [The identification of -workers . Dkk2 ano et al. . We haveWnt1 and TCF1, were not found in the current study. Their absence, however, is not surprising. Wnt1 expression is observed during early development and its activity is important in determining the fate of differentiating cells [Wnt1 activity may be of limited significance. TCF1 expression is mostly restricted to cells of T-cell lineage [Genes, including ng cells ,37. In d lineage -40 and tsFRP1 has been shown to induce IOP elevation in mice [2O2 treatment (The role of acute oxidative damage in modulating Wnt signaling in TM cells was also assessed. Two proteins, namely β-catenin and sFRP1, were chosen for western blot analysis. The former is a key element in Wnt/β-catenin signaling, and numerous studies in the literature examined the β-catenin level as a measure of Wnt activation ,41. The  in mice . Our resreatment . FurtherIn the present study, the effects of oxidative stress on sFRP1 and β-catenin protein levels were evaluated for time points up to 4 h. In the literature, changes in β-catenin and sFRP1 protein levels have been shown to arise within a relatively short period of time. For instance, Aberle and co-workers  detected2O2 for 20 min were reported to yield a modest but significant activation of TCF [2O2 exposure [2O2 suppressed TCF activity in murine skeletal muscle C2C12 myoblast [2O2 effect may be concentration, cell-type, and/or context dependent. The 1 mM, 30 min-H2O2 treatment in our experiments had little impact on Wnt activity (Previously, NIH3T3 and HEK293 cells treated with 300 μM Hn of TCF . Accumulexposure . Howeverexposure ,19. It wmyoblast , osteoblmyoblast , and HEKactivity , but it activity , as was In conclusion, the current study revealed the existence of many Wnt elements that had not been previously tested or reported and demonstrated the presence of canonical as well as noncanonical Wnt components in human TM cells. Our results in addition indicated that the protein levels of two key Wnt signaling components, β-catenin and sFRP1, are unmodified by oxidative stress, a possible causal factor thought to be involved in glaucoma. This suggests that Wnt signaling may not be a factor related to oxidative damage in the TM system.


Furthermore, the effect of these genetic alterations on the transcriptional activity of the β-catenin/E-cadherin adhesion complex is not yet known. Within this context, we investigated the possible association of β-catenin mutations in exon 3 with the β-catenin/E-cadherin adhesion complex transcriptional activity by determining their mRNA levels and the levels of E-cadherin protein expression in a subset of these carcinomas.A total of 63 sporadic colorectal adenocarcinomas and adjacent normal tissues at least 2 cm from the distal negative margin of the tumour were obtained from surgically treated patients at Nikaia Hospital, Athens, Greece. None of the patients received irradiation preoperatively.β-catenin nucleotide substitutions as previously described  H-108  in formalin-fixed paraffin-embedded tissue sections from the same subset of 37 SCRCs as previously described  are shown inexamined . Similarexamined . These rβ-catenin mRNA in 24 out of 37 (65%) of the carcinomas examined  and 23out of 37 (62%), respectively , APC, and Axin, while in the nucleus binding to TCF leads to promotion of gene transcription , these results point out an additional possible physiological association between E-cadherin expression and the β-catenin signalling pathways described above. However, it is not yet known whether changes in E-cadherin mRNA and/or protein expression levels may affect β-catenin signalling and our study cannot rule out the possibility of cytoplasmic β-catenin stabilisation at the protein level.A paralleled decreased expression of E-cadherin mRNA was demonstrated in 25 out of 37 of the carcinomas (68%), and this was followed by a comparable decrease of E-cadherin IHC expression (grade I) in 21 out of 37 (57%) of SCRCs . E-cadheβ-catenin and E-cadherin gene expression were significantly associated with the presence of lymph node metastases (LN) (P=0.018) in 20 out of 37 (54%) and in 22 out of 37 (59%) (P=0.001) of the carcinomas examined, respectively. Similarly, a significant inverse association was observed between decreased E-cadherin IHC detection (grade I) and LN presence (P=0.012) in 20 out of 37 (54%) of these carcinomas. The demonstrated impairment of β-catenin/E-cadherin adhesion and its association with LN metastasis (β-catenin system through multiple mechanisms and the subsequent suppression of cell-cell adhesiveness may trigger the release of cancer cells from the primary tumour foci and confer invasive properties on the primary tumour mass.Both decreased astasis  suggestsβ-catenin exon 3 are an infrequent event in SCRCs and that β-catenin genetic alterations are not responsible for the demonstrated loss of β-catenin/E-cadherin transcriptional activity.In essence, this study shows that the mutations within


Insights into how the Frizzled/LRP6 receptor complex receives, transduces and terminates Wnt signals will enhance our understanding of the control of the Wnt/ß-catenin pathway.Drosophila cells expressing an activated form of LRP6 and a β-catenin-responsive reporter. This screen resulted in the identification of Bili, a Band4.1-domain containing protein, as a negative regulator of Wnt/β-catenin signaling. We found that the expression of Bili in Drosophila embryos and larval imaginal discs significantly overlaps with the expression of Wingless (Wg), the Drosophila Wnt ortholog, which is consistent with a potential function for Bili in the Wg pathway. We then tested the functions of Bili in both invertebrate and vertebrate animal model systems. Loss-of-function studies in Drosophila and zebrafish embryos, as well as human cultured cells, demonstrate that Bili is an evolutionarily conserved antagonist of Wnt/β-catenin signaling. Mechanistically, we found that Bili exerts its antagonistic effects by inhibiting the recruitment of AXIN to LRP6 required during pathway activation.In pursuit of such insights, we performed a genome-wide RNAi screen in These studies identify Bili as an evolutionarily conserved negative regulator of the Wnt/β-catenin pathway. Wnt signaling through β-catenin regulates the expression of genes involved in cell proliferation and cell fate during development, in adult homoeostasis, and in diverse diseases Signaling events immediately following Wnt binding to the receptor complex are poorly understood. Previous studies have shown that in the presence of Wnt ligand, Dishevelled (DVL) facilitates phosphorylation of the cytoplasmic tail of LRP5/6 Drosophila cells to identify genes that regulate LRP6 mediated activation of a β-catenin reporter. Here we describe Bili (Band4.1 inhibitor LRP interactor), a previously uncharacterized FERM domain containing protein. Analyses of Bili function in Drosophila, zebrafish, and cultured human cells support the conclusion that Bili is an evolutionarily conserved antagonist of β-catenin signaling. Mechanistically, we show that Bili regulates the recruitment of AXIN to LRP6.We performed a genome-wide RNAi screen in Drosophila mutagenesis screens have resulted in the identification of several core Wg signaling proteins Drosophila dsRNA screen clone 8 (cl8) Drosophila cells were transfected in a 384-well plate format with individual dsRNAs, dTF12, ΔNLRP6 and a Renilla luciferase control for cell viability and transfection efficiency. The cells were incubated for four days to allow knockdown of target RNAs and normalized reporter luminescence was then measured as an indicator of Wnt pathway activity. Approximately 0.5% of the library (∼100 dsRNAs) modified the activation of the reporter by ΔNLRP6 in a manner validated by low throughput confirmation of the primary screen. dsRNA targeting eighteen genes with known human orthologs increased reporter activation two-fold or greater  database were not pursued further. greater . Seven oFRMD8/FKSG44 (human), also has orthologs in worms, zebrafish and mice (Ezrin Radixin Meosin) are highly conserved protein domains important for directing plasma membrane association and, in some cases, linking integral membrane proteins to the actin cytoskeleton and modulating signaling Of the 11 remaining candidate negative regulators, we chose to focus on CG11848 (dBili) due to its predicted plasma membrane localization and the specificity of its effect on the Wg pathway. More specifically, of the 12 other screens in the DRSC database, dBili was identified only as a weak hit in a MAPK kinase screen . Bili, annotated as CG11848 (fly) and and mice . In humaand mice . Bili isand mice . FERM doWg-dependent reporter activation  in the paired domain (prdGAL4>UAS-shdBili). Quantitative real time-PCR (qRT-PCR) analysis following expression of dBili shRNA under the control of prd-GAL4 in the  embryos . As a coo change . Importao change . Furtherripe 2/3 . Thus, dengrailed (En) and defines para-segment boundaries Drosophila embryonic development.Next we assessed the molecular consequences of depleting dBili in the paired domain. During embryonic segmentation, Wg activates and maintains the expression of The Wg signaling pathway is highly conserved throughout evolution. Therefore, we next asked if Bili negatively regulates Wnt signaling in vertebrates. To this end, we asked if Bili function is conserved in zebrafish. Ectopic activation of Wnt/β-catenin signaling in early zebrafish development causes dose-dependent anterior truncations and mesodermal defects  [17]. IfWe next investigated the effect of zfBili gain-of-function on zebrafish development. Embryos injected with zfBili RNA, but not control RNA, displayed a slight expansion of anterior structures, which is consistent with diminished Wnt/β-catenin signaling (data not shown) Renilla luciferase normalization control. Activation of the reporter in RKO cells following treatment with WNT3A conditioned media was enhanced by transfection of either hBili or AXIN1 siRNA and repressed by β-catenin siRNA  cells expressing a β-catenin responsive luciferase reporter and in siRNA . Similarin siRNA . Consistin siRNA .AXIN2 , a GSK3 inhibitor We next carried out epistasis experiments in HEK293T cells expressing a β-catenin responsive luciferase reporter and Based on these results we predicted that Bili might regulate signaling from the Wnt co-receptors LRP5/6 or Frizzled. In direct support of this prediction, tagged hBili colocalizes with tagged LRP6 as detected by immunocytochemistry (ICC) . FurtherDuring Wnt signaling, LRP6 is phosphorylated in a Wnt-dependent manner by CSNK1G1 and GSK3B, which then facilitates the binding of AXIN to LRP6 To address this hypothesis we employed co-immunoprecipitation to monitor the association of AXIN and LRP6 following gain-of-function  or loss-Many aspects of Wnt/β-catenin signal transduction remain poorly understood. One aspect in particular is the transduction events occurring at the plasma membrane following Wnt activation of the Frizzled/LRP6 coreceptor complex. Studies over the last several years have shed light on these events by uncovering several kinases that phosphorylate LRP6 resulting in its recruitment of AXIN We propose that inhibitors of the Wnt/β-catenin pathway fall into two groups: ‘constitutive inhibitors’ that keep basal levels of β-catenin low, such as members of the degradation complex; and ‘inducible inhibitors’ that act outside of the degradation complex and may function only when the pathway has been activated. The ‘constitutive inhibitors’ would include APC and AXIN, while ‘inducible inhibitors’ would include NKD and Bili. Consistent with this categorization, NKD and Bili both antagonize a WNT signal between receptor activation and the degradation machinery as well as having similar phenotypes in both flies The screen was performed as previously described Renilla luciferase (10–40 ng) for normalization. FOPFlash and fuBAR, which contain mutated response elements and do not respond to WNT/β-catenin signaling, were used as control reporters. We used RKO cells that stably express BAR and RenillaWNT1 (0.1 ng) and TOPFLASH (1 ng)/Renilla luciferase(10 ng)). Lysis and luminescence  was carried out 24 h after WNT treatment. For GOF experiments, cells were seeded to 50% confluency after which cDNA (GFP (50–100 ng) or Bili-FLAG (25–100 ng), TOPFLASH, Renilla luciferase, WNT1 (0.1 ng), ΔNLRP6 (5 ng), DVL (5 ng), β-catenin (1 ng) was transfected. BIO (0.5 µM) was also added to the cells at this point. Cells were lysed and luminescence measured 24 h hours later. Bili siRNAs were purchased from Ambion; siRNA ID# 332724 that targeted Exon 8  and siRNA ID# 45515 that targeted Exon 7, 8. Control siRNA and Axin 1 & 2 siRNAs have been described β-catenin reporter assays were carried out in the 24-well or 48-well plate format in HEK293T cells transiently expressing SuperTOPFLASH (1–5 ng) or BAR (β-catenin Activated Reporter) and Drosophila Protocols” by William Sullivan et al., CHSL press.In situ hybridization and immuno-staining in the fly embryos and larval imaginal discs were performed using standard protocols described in the laboratory manual “GAA GAT CTC CCA ATC GGC GAA CAA TTC ATA CC – 3′; Act Reverse 5′ - GAA GAT CTT TGA ACG CGA CTT GAG AGC GG – 3′; Armadillo: Arm Forward 5′ – CAA CGT TCC TCG ATA GCC AC – 3′; Arm Reverse 5′ – CGA ATG AGT GCC CGG TTG TG – 3′; C11848/dBili: CG11848 Forward 5′ – CTT GGC TGT GGC TAC CGA TC - 3′; CG11848 Reverse 5′ – GCG CCA TAG AAT GGC AGA GC – 3′.RNA was isolated from Drosophila embryos using Trizol and was further purified using the RNA “clean-up” protocol with the Qiagen RNAeasy kit. This was followed by reverse transcription (Applied Biosystems - High Capacity cDNA Reverse Transcription Kit) and quantitative PCR (SYBR green) using Applied Biosystems 7300 Real-Time PCR Machine. PCR primers used were as follows: Actin: Act Forward 5′ – 5′ GTAGTAAAGCTTGCCACCATGGCCCTGAGGATGGACGGG 3′ and reverse primer sequence 5′ TACTACGTCGACTCAGCCCTGCTCCAGGCTGT 3′. Zebrafish Bili was cloned out of a mixed embryonic library (6 hpf-24 hpf) using forward primer 5′ TCTCCAGCTCAGGATTTGTTGGTG 3′ and reverse primer 5′ TACTACCTCGAGTCAACTTTCAGTCAC 3′ based on an ENSEMBLE BLAST prediction using Bili. Since the ATG was missing, a 5′ RACE kit (Invitrogen) was used with a nested primer 5′ GCCACTGACGACACAGCTTGTAA 3′ that yielded the full-length clone.MGC clone for human Bili (MHS1010-9204707) was purchased (Open biosystems). Bili was sub-cloned into pCS2+ using the EcoR1 and Xho1 sites. Forward primer sequence AXIN-MYC (300 ng), GSK3B (50 ng) and Bili-FLAG (200 ng). Cells were lysed the next day in a buffer containing 1% Triton-X, 50 mM Tris-HCL pH 7, 150 mM NaCl, protease and phosphatase inhibitors. LRP6-EGFP was immunoprecipitated using polyclonal GFP and protein G beads for 2 hours. The beads were washed three times with the lysis buffer that contained 0.6 M NaCl instead of 150 mM NaCl. Proteins that bound were eluted using SDS gel loading buffer and western blot analysis was used to detect GFP, MYC, FLAG and GSK3B. Quantification was done using NIH image.HEK293T cells were made to stably express LRP6-EGFP by transfecting pCS2+LRP-EGFP and pPUR (Clontech) into cells and treating with 2 mg/ml of puroMYCin. Stable cells were seeded into 6-well plates at 50% confluency and transfected with Drosophila S2R+ cells were used for coIP. S2R+ cells were co-transfected with HA-tagged dBili and Myc-tagged,ΔNLRP6 IP was performed with Rabbit anti-Myc antibody  and IB with both anti-Myc (1∶500) and anti-HA . Standard IP protocol was used with mild lysis buffer and Protein A/G sepharose.In situ hybridization and microinjections were carried out as previously described on zebrafish online resource, ZFIN. ATG blocking morpholino sequences (Gene Tools) GGAAGTCGCCATCATCTCCCTCCAT (bilimo) and GAGACACTCTCCTTCGATTCAGAAG (bilimo2). A 10 µM stock was diluted to 1.5 µM. bilimo, bilimo2, or control morpholino at this concentration were then coinjected into 1-cell embryos at three doses of wnt8 RNA . The same wnt doses were used for zfBili overexpression coinjection experiments with Renilla as a control at 50 ng/µl stock concentration. Injection drop size 0.5–1 nl.5′CCAAGCAGGCCGAACT3′, Bili reverse 5′CCTTGCCGTCCTCCACGTA3′, GAPDH forward 5′CCA CCC ATG GCA AAT TCC ATG GCA3′, GAPDH reverse 5′TCTAGACGGCAGGTCAGGTCCACC3′. Axin 2 forward 5′CTCCCCACCTTGAATGAAGA3′ Axin 2 reverse 5′ TGGCTGGTGCAAAGACATAG3′RNA was isolated following treatment using Qiagen RNAeasy kit. This was followed by reverse transcription (Invitrogen ThermoScript or SuperScript) and quantitative PCR (sybr green) using Roche Light cycler 2.0. PCR primers Bili forward NM_143085 (Drosophila), XM_682750.2 (Zebrafish), NM_031904 (Human).Table S1Table showing dsRNA targeting eighteen genes with known human orthologs that increased reporter activation two-fold or greater.(0.01 MB DOC)Click here for additional data file.Figure S1(A) Sequence alignment of Bili protein shows it is well conserved phylogenically. (B) Human Multi Tissue Northern blot show mRNA expression. (C) Illustration of Bili protein structure and domains.(2.14 MB EPS)Click here for additional data file.Figure S2(A) qRT-PCR analysis of dBili knockdown. Expression of short-hairpin (shRNA) dbili (#3M) under the control of prd-GAL4 in the Drosophila embryo results in >75% knockdown in message level (Red bar) as compared to prd-GAL4 control embryos (Blue bar). As a control, Armadillo mRNA was not affected in embryos expressing dBili shRNA. (B) Embryos expressing dBili shRNA under the control of even skipped stripe 3/7-GAL4: These embryos display a partial lack/disruption of the 2nd or 3rd denticle belt which coincides with the eve-stripe 3 expression. This phenotype is consistent with a localized increase in Wingless signaling activity in the region around eve-stripe 3. The same however was not observed for stripe 7 (posterior end of the embryo), perhaps due to differential expression of GAL4 in stripe 3 versus stripe 7.(3.11 MB EPS)Click here for additional data file.Figure S35′-GTAATACGACTCACTATAGGGAGA GAAGATACAAGTGAGGCATTC-3′ and Reverse primer: 5′GTAATACGACTCACTATAGGGAGAGGCAAATAAAATATCTGATGGGTGCGTGG-3′ (B) Knockdown of dBili with ds11848_2 displayed a modest increase (∼35–40%) in dTF12 (Wg-reporter) activity. (C & D) Expression of dBili-HA strongly inhibits dTF12-reporter activity when the pathway is activated by Wg overexpression. The inhibitory effect of dBili is comparable to that of Axin expression in both S2R+ (C) and clone8 (D) cells. (E) dBili protein (in GREEN) is localized in a concentric ring, just inside and abutting Arm protein (in RED) at the membrane of cells in the drosophila embryo. (E') Magnitifed view of boxed region in panel E.(A) Knockdown of dBili with a second dsRNA, ds11848_2, revealed only ∼30% knockdown of endogenous message compared to the original dsRNA (DRSC1.0-CG11848) which robustly knocked down message levels by >50%. Primer sequence used for the generation of ds11848_2 PCR is as follows: Forward primer: (5.47 MB EPS)Click here for additional data file.Figure S4The zebrafish homolog of Bili (zfBili) is expressed during embryogenesis. In situ hybridization using a sense (left column) and antisense probes (right column) were used to detect mRNA. (A) zfBili is expressed maternally as it is detected in the animal pole 4 h post-fertilization. (B) zfBili continues to be expressed ubiquitously at 50% epiboly. (C) 24 hpf zfBili remains ubiquitous but shows specific staining in the otic vesicle (arrow head). (D) Weak Bili expression in the tail at 36 hpf. (E) A second morpholino targeting zfBili (bilimo2) but not a control morpholino (como) enhances the Wnt8 overexpression phenotype (n = 50 for each condition). Embryos were scored as wt (green), small eyes (yellow), no eyes (orange) or severe (red). Data shown is representative from four independent experiments.(10.82 MB EPS)Click here for additional data file.Figure S5Bili negatively regulates Wnt/{capital β-catenin signaling in mammalian cells. (A) siRNA mediated knockdown of hBili enhances Wnt1 mediated induction of a β-catenin responsive reporter. HEK293T cells were transfected with siRNA targeting control, β-catenin, or hBili. Cells were then transfected with WNT1 cDNA and a β-catenin responsive reporter and assayed the following day. (B) hBili siRNA effectively knocked down hBili transcripts in HEK293T as measured by qRT-PCR. (C) hBili siRNA effectively decreased the expression of a hBili-Venus fusion protein as assayed by fluorescence measurement. hBili siRNA had no effect on Venus expression. (D) A second siRNA (Bili #2) targeting hBili enhanced WNT1 mediated activation of a β-catenin responsive reporter. (E) Bili #2 siRNA also effectively decrease expression of a hBili-Venus fusion protein with no effect on Venus alone. (F) hBili knockdown in HEK293T cells with hBili siRNA enhanced the WNT3A mediated transcriptional induction of endogenous AXIN2 transcripts as assayed by qRT-PCR. Error bars represent STDEV .(1.48 MB EPS)Click here for additional data file.Figure S6Bili does not affect control reporters or CREB responsive reporters. (A) HEK293T cells were transfected with FUBAR, a control reporter not responsive to Wnt/β-catenin signaling, renilla luciferase normalization, and control or hBili siRNA. FUBAR was not responsive to WNT3A conditioned media (right two bars) and hBili siRNA had no effect. (B) HEK293T cells were transfected with FOPFlash, another control reporter not responsive to Wnt/β-catenin signaling, renilla luciferase normalization, and GFP cDNA or increasing doses of hBili cDNA or control siRNA or hBili siRNA. Neither hBili cDNA control nor hBili siRNA had an effect on the reporter. (C) hBili siRNA HEK293T cells stably expressing a CREB responsive reporter were transfected with renilla luciferase normalization, and control, hBili, or hBili#2 siRNA and treated with DMSO, 1 uM forskolin, or 10 uM forskolin. Error bars represent STDEV.(1.14 MB EPS)Click here for additional data file.