Recent experiments have explored effects of activities of kinases other than the well-studied GSK3β, in wnt pathway signaling, particularly at the level of β-catenin. It has also been found that the kinase PKA attenuates β-catenin degradation. However, the effects of these kinases on the level and degradation of β-catenin and the resulting downstream transcription activity remain to be clarified. Furthermore, the effect of GSK3β phosphorylation on the β-catenin level has not been examined computationally. In the present study, the effects of phosphorylation of GSK3β and of phosphorylations and degradation of β-catenin on the kinetics of the wnt signaling pathway were examined computationally.The well-known computational Lee-Heinrich kinetic model of the wnt pathway was modified to include these effects. The rate laws of reactions in the modified model were solved numerically to examine these effects on β-catenin level.The computations showed that the β-catenin level is almost linearly proportional to the phosphorylation activity of GSK3β. The dependence of β-catenin level on the phosphorylation and degradation of free β-catenin and downstream TCF activity can be analyzed with an approximate, simple function of kinetic parameters for added reaction steps associated with effects examined, rationalizing the experimental results.The phosphorylations of β-catenin by kinases other than GSK3β involve free unphorphorylated β-catenin rather than GSK3β-phosphorylated β-catenin*. In order to account for the observed enhancement of TCF activity, the β-catenin dephosphorylation step is essential, and the kinetic parameters of β-catenin phosphorylation and degradation need to meet a condition described in the main text. These findings should be useful for future experiments. Briefly, wnt signaling enhances the level of the output signal protein, unphosphorylated β-catenin, which then binds with TCF to induce associated gene expression in the nucleus. At steady state (SS), the level of β-catenin is balanced by its synthesis and degradation. The so-called destruction cycle is a major mechanism of degradation, in which phosphorylation of β-catenin by GSK3b is a key step [The Wnt/β-catenin signaling pathway (named wnt pathway hereafter for simplicity) plays a significant role in cell proliferation, differentiation, and apoptosis. These have implications for aspects of cell development, stem cells and cancer . Many chkey step . When thRecent advances have further illustrate how phosphorylation and dephosphorylation of major components in the wnt pathway affect the stability of β-catenin and its TCF transcription activity -7. It haIn addition to experimental studies, computational studies have also aided understanding of the dynamical behaviors of this pathway and how it interacts with other pathways ,10-13. TIn addition to the Lee-Heinrich model of the wnt pathway itself, this model has been used and extended to examine the effect of Apc mutations on the wnt signaling pathway , cross-tIn the present study, in the light of the recent experimental studies described above -7, I aimThe COPASI program  was usedG/kG' and kβ/kβ', respectively. The rate constant of β-catenin degradation is denoted kβdeg. For simplicity, these kinetic parameters were all set equal to 1 min-1 except in some cases (see Section III). The parameters of interest were then varied to examine their effects on the kinetics of the wnt pathway. Initial concentrations were values of SS concentrations used in the Lee-Heinrich model [+, β-catenin+, and β-catenin*+, with initial concentrations all set at zero. The superscript * denotes phosphorylation by GSK3β. Other phosphorylations are denoted by the subscript+. The differential equations were solved for 20000 minutes, or as long as 40000 minutes, to ensure they reached SS.The forward/backward reaction rate constants of GSK3β and β-catenin phosphorylations are denoted kch model  with addTo examine how selected parameters affect the SS concentration of β-catenin in a modified model, control coefficients were calculated as well. These were defined by cat is concentration of β-catenin and k is the parameter of interest. This coefficient was calculated numerically by varying the associated parameters by 1% and solving the kinetic equations over enough time to obtain SS concentrations, in order to calculate control coefficients as in [where β-ts as in . Finallyts as in . InitialInitially, I examined these two effects without including the β-catenin degradation step  (when kdeg βand kβ were varied due to the change of PKA activity), needs to dominate over k13 so that increased inhibition of degradation can enhance TCF activity. A computed result is shown in Figure respectively. The computations for the added reaction steps taking place at β-catenin*kβ values, which make the term kβ/(1+kβ'/kdegβ) smaller or larger than k13, were carried out as well. When kβ was equal to or 1 order smaller than k13, the level of un-phosphorylated β-catenin did not change significantly when the β-catenin+ degradation constant, kdegβ, was varied  gradually dominates over k13. The kβ value was increased up to 1.0. In the range of 0.5–1.0, a significant change in the un-phosphorylated β-catenin level due to varying kdeg βwas seen, as shown in Figure + affect the added reactions at β-catenin+ and the level of un-phosphorylated β-catenin, computations with both added reaction parts present in the model were also carried out. Variations of both degradation rate constants in the added reaction steps, which may change the level of β-catenin significantly, showed that the computed results are similar to those obtained with each part computed separately in the model as shown in Figure -4 and 10-2 nM scales, respectively, the level of un-phosphorylated β-catenin  is roughly 4 and 1–2 orders higher than the APC*/Axin*/GSK3β/β-catenin complex and the downstream phosphorylated β-catenin, β-catenin*, respectively. Therefore, the presence of the added reaction steps at β-catenin*+ has no significant effect on the SS level of components in the added reaction step at β-catenin+ or the un-phosphorylated β-catenin. The added reaction steps at β-catenin+ alone can describe the parameter dependence of β-catenin level due to non-GSK3β phosphorylation, the succeeding degradation, and the resulting TCF activity. Finally, to verify the findings in the present computational study, it is suggested that the following be investigated in future experiments: (1) to seek the phosphatase of β-catenin+ and obtain its kinetic rate constant, kβ'. (2) to obtain the kinetic constants, kβ and kdegβ, of the added reaction steps 19 and 20, respectively, associated with non-GSK3β phosphorylation of β-catenin. In the long-run, the model will be extended to include more unexamined effects caused by other components in the course of wnt signaling pathway research in order to draw a more complete picture.To test this analysis further, computations with varied The present computational study gave the following results: (1) Phosphorylation of GSK3β increases the level of β-catenin in an approximately linear fashion. (2) β-catenin non-GSK3β phosphorylation is more likely to take place at free unphorphorylated β-catenin rather than GSK3β-phosphorylated β-catenin*, thus accounting for the observed enhancement of TCF activity. (3) To account for the effects of kinases such as PKA, which phosphorylates β-catenin and inhibits β-catenin degradation at the same time, the dephosphorylation step is essential. In addition, the kinetic parameters of the reactions taking place at the unphosphorylated β-catenin site need to meet the conditions described above in describing the simplified mathematical functions (1) and (2). Finally, the qualitative behaviors found in the present study should be useful for future experiments in charactering how the effects of GSK3β phosphorylation, β-catenin non-GSK3β phosphorylations, and β-catenin degradation, affect the kinetics of the wnt pathway, and can be verified in future experiments.GSK3: Glycogen synthase kinase 3; PKA: Protein kinase A; TCF: T-cell factor.The author declares that they have no competing interests.


Wnt4 or β-catenin was inactivated in the fetal ovary, female germ cells underwent degeneration. In this study, we set out to understand whether these two factors belong to the same pathway and how they maintain female germ cell survival. We found that activation of β-catenin in somatic cells in the Wnt4 knockout ovary restored germ cell numbers, placing β-catenin downstream of WNT4. In the absence of Wnt4 or β-catenin, female germ cells entered meiosis properly; however, they underwent apoptosis afterwards. Activin βB (Inhbb), a subunit of activins, was upregulated in the Wnt4 and β-catenin knockout ovaries, suggesting that Inhbb could be the cause for the loss of female germ cells, which are positive for activin receptors. Indeed, removal of Inhbb in the Wnt4 knockout ovaries prevented female germ cells from undergoing degeneration. We conclude that WNT4 maintains female germ cell survival by inhibiting Inhbb expression via β-catenin in the somatic cells. Maintenance of female germ cells hinge upon a delicate balance between positive (WNT4 and β-catenin) and negative (activin βB) regulators derived from the somatic cells in the fetal ovary.Female germ cells are essential for organogenesis of the ovary; without them, ovarian follicles do not form and functional and structural characteristics of the ovary are lost. We and others showed previously that when either  Dazla and Factor in the germline α  led to degeneration of oocytes and failure in folliculogenesis Female germ cells not only are essential for the propagation of species but also play an important role in ovarian organogenesis. Presence of germ cells is required for the formation of follicles, the functional unit of the ovary. Disruption of germline-specific genes, such as Wnt) family of genes, including Wnt4, Wnt5a, Wnt6, and Wnt9a, are found expressed in the somatic cells of the fetal ovary Wnt4 or β-catenin resulted in degeneration of female germ cells starting at 16.5 dpc Once they have migrated to the gonad, germ cells start to differentiate by following their intrinsic programs as well as responding to instructions from the somatic environment fCtnnb1/−) loxP sties that flank the β-catenin gene, therefore producing a null allele of β-catenin. Inactivation of β-catenin in the SF1-positive somatic cells of fetal ovaries resulted in a progressive loss of female germ cells starting at 16.5 dpc f/+SF1/cre; Ctnnb1 or f/−Ctnnb1) starting at 17.5 dpc  mouse by introducing the Steroidogenic factor 1-cre (SF1/cre) transgene into an embryo carrying floxed and null β-catenin alleles Ctnnb1) specifically in the SF1-positive somatic cells in the Wnt4 KO ovary. fl.(ex3)Ctnnb1 mice contain a genetically engineered β-catenin gene that loxP sequences are inserted in either side of the exon 3. The peptide encoded by the exon 3 is responsible for degradation of β-catenin. Once the exon 3 is removed by the Cre recombinase, the mutant β-catenin becomes resistant to degradation and therefore constitutively active in the SF1-positive somatic cells The germ cell loss phenotype in the β-catenin cKO ovary shares striking similarities with that in the Wnt4 knockout ovaries should restore normal germ cell development. We examined the total germ cell numbers in the newborn ovaries from controls ), Wnt4 KO , and Wnt4 KO plus active β-catenin ; Wnt4 KO ovary was significantly lower than the controls Wnt4 ovary  ovary , indicatWnt4 or β-catenin resulted in ectopic appearance of androgen-producing CYP17-positive cells in the ovary  was significantly elevated in the Wnt4 KO ovaries Wnt4 KO ovary decreased Inhbb mRNA expression to the level similar to that in the control  and negative regulators  expression was not altered Wnt4 expression via β-catenin.Somatic cells of the fetal ovary are the supporting cells that nurture the germ cells and provide them proper environment to grow. Ovaries with active β-catenin in the somatic cells are larger than the controls, suggesting that the activation of β-catenin promotes proliferation of somatic cells. WNT/β-catenin signaling pathway is known to regulate genes that are involved in cell proliferation and cell fate decision during embryogenesis Wnt4 KO and β-catenin cKO fetal ovaries after anti-flutamide treatment, therefore excluding the involvement of androgens. Furthermore, androgen receptors are not present in germ cells at fetal stages Wnt4 KO and β-catenin cKO fetal ovaries. Rescue of the germ cell loss phenotype in the −/−Wnt4; −/−Inhbb double KO ovary provides a genetic link implicating Inhbb as the gene responsible for germ cell demise. Inhbb encodes the subunit for inhibin B and activin B. Germ cells are known to express receptors (Acrr-IB and ActR-IIB) for activins Wnt4 and β–catenin could therefore act directly on female germ cells and cause their death. Using Transcription Element Search System (TESS) program, we found several putative β-catenin LEF/TCF response elements in the promoter region of Inhbb. β-catenin could bind to these response elements and suppress the expression of Inhbb in the fetal ovary.Germ cell loss still occurred in Fst) knockout fetal ovaries Fst acts downstream of WNT4 Fst is dependent upon a consensus β-catenin LEF/TCF binding site in the promoter region Fst was lost in the β-catenin cKO ovary Fst downstream of β-catenin. FST is known to bind activins with high affinity, therefore preventing activins from activating their receptors Inhbb mRNA is present in mouse gonads of both sexes at 11.5 dpc. Its expression is down-regulated but remains detectable in the ovary at 12.5 dpc Wnt4 and β-catenin cKO ovary where Inhbb expression is upregulated, Inhbb mRNA expression levels are not altered in the absence of FstInhbb and by activating FST to antagonize activin B protein  and Activin B (the protein product of Inhbb) is critical for the maintenance of female germ cells during embryonic stage. It is possible that increasing germ cell apoptosis close to birth is the result of a shifted balance toward action of activin B. If our hypothesis is correct, one would predict that loss of Inhbb should lead to decrease in germ cell apoptosis and presumably more oocytes in the ovary. Inhbb knockout females are fertile despite an increase in length of gestation and a decrease in ability of nursing Inhbb knockout ovary.It is known that oocytes start entering apoptosis prenatally, therefore leaving a finite number of oocytes for the rest of the reproductive life of female individuals. At least two hypotheses have been proposed for the cause of female germ cell demise during embryogenesis Wnt4 or β-catenin is inactivated, female germ cells enter meiosis and progress to meiosis prophase I normally, based on the time course analysis of chromosome smears of germ cells and examination of expression of SCP3 and γH2AX Wnt4/β-catenin. Studies on the R-spondin1 (Rspo1) KO mice show that Rspo1 is the upstream regulator of WNT4 and β-catenin in ovarian development Rspo1, components of the RA pathway are not significantly affected Rspo1 KO ovary at 14.5 and 16.5 dpc, germ cell entry into meiosis appeared to be normal based on the SCP3 staining When either Drosophila and mouse. Activation of β-catenin in PGCs promotes proliferation in Drosophila whereas it delays cell cycle progression in mouse Involvement of WNT/β-catenin pathway in regulating proliferation of primordial germ cell (PGCs) is evident in All procedures described were reviewed and approved by the Institutional Animal Care and Use Committee at University of Illinois, and were performed in accordance with the Guiding principles for the Care and Use of Laboratory Animals.floxed/−SF1/cre;Ctnnb1 embryos were derived from breeding of the +/−Ctnnb1; SF1/cre, which carrying Cre recombinase under the control of the Sf1 promoter and its regulatory elements floxed/floxedCtnnb1 parental strains. +/−Wnt4 mice were obtained from Jackson Laboratory (strain 129-Wnt4tm1Amc/J). fl(ex3)Ctnnb1 mice were obtained from Harada et al. Wnt4 knockout ovary, +/−; SF-1/creWnt4 were mated with +/−Wnt4; fl(ex3)Ctnnb1 mice to obtain −/−;Sf-1cre; Ctnnb1fl(ex3)Wnt4 embryos. To obtain the Wnt4 and Activin β B double knockout mice , +/−Wnt4 mice were mated to +/−Inhbb mice to generate +/−Wnt4; +/−Inhbb double heterozygotes. +Wnt4/−; +/−Inhbb double heterozygotes were mated to generate −/−Wnt4; −/−Inhbb double knockout mice. The day when the vaginal plug was detected in the mated female was considered as 0.5 dpc. Genotypes were determined by PCR using gene specific primers. The primers were: SF-1/Cre genotyping: 5′-GTGTGAACGAACCTGGTCGA AATCAGTGCG-3′ and 5′-GCATTACCGGTCGATGCAACGAGTGATGAG-3′; Ctnnb null allele: 5′-AATCACAGGGACTTCCATACCAG-3′ and 5′-GCCCAGCCTTAGCCCAACT-3′; Ctnnb1 wild type and floxed alleles: 5′-AAGGTAGAGTG ATGAAAGTTGTT-3′ and 5′-CACCATTGTCCTCTGTCTATTC -3′; Wnt4 null and wild type alleles: 5′- CTG AGGAAGAGCAGGGTCAC -3′, 5-ATGGTCACC CCCATTTTACA -3′ and 5-TGGATGTGGAATGTGTGCGAG-3′; fl(ex3)Ctnnb1 alleles 5′-GGTAGTGGTCCCTGCCCTTGA CAC-3′ and 5′-CTAAGCTTGGCT GGACGTAAACTC-3′; Inhbb null allele: 5′-CTTGGGTGGAGAGGC TATTC-3′ and 5′ - AAAGCTGATGATCTCGGAGACG - 3′; Inhbb wild type allele: 5′-ATGGTC ACGGCC CTG CGC AA-3′ and 5′-GCGGAT CCC TCT GCA AAG CTG ATG ATT TC-3′; Sry genotyping: 5′-TGAAGCTTTTGGCTTTGAG-3′ and 5′-CCGCTGCCAAATTCTTTGG-3′. All experiments were performed on at least two to five animals for each genotype.Samples were fixed in 4% paraformaldehyde overnight at 4°C and then washed in PBS for 5 minutes (3 times) Samples were put through a sucrose gradient  and incubated in 1∶1 20% sucrose and OCT freezing media (Tissue-Tek) overnight at 4°C. Samples were embedded in 1∶3 20% sucrose and OCT mix and cut to 10 µm thick frozen sections. Sections were washed with PBS and then blocked in the blocking solution (5% heat-inactivated donkey serum and 0.1% Triton X-100 in PBS) for 1 h at room temperature. Primary antibodies were added to the blocking solution and incubated with sections at 4°C overnight. Sections were then washed with the washing solution (1% heat inactivated donkey serum and 0.1% Triton X-100 in PBS) followed by incubation in the blocking solution with the corresponding secondary anybodies. Sections were then washed with the washing solution and mounted with DAPI antifade reagent. The sources and dilution of primary antibodies were the rat monoclonal antibody against germ cell nuclear fraction , the rabbit polyclonal antibody against cleaved caspases-3 , and the rabbit polyclonal antibody against CYP17 . All the secondary antibodies were purchased from Jackson Immunochemical and a 1∶200 dilution was used.Fetal germ cell chromosome smear and immunostaining were performed according to the protocol described in For immunostaining of spread chromatin, slides were washed three times (10 min each) in 10% antibody dilution buffer . Then slides were incubated with anti-SCP3 antibody  in a humid chamber overnight at 4°C. Samples were washed three times (10 min each) in 10% antibody dilution buffer. Slides were then incubated with secondary antibody for 2 h at room temperature in the dark followed by three washes (5 min each) in PBS. Slides were air-dried and mounted with DAPI antifade reagent.Newborn ovaries were obtained from 3 animals for each genotype. Samples were fixed in 4% paraformaldehyde in PBS at 4°C and processed according to the immunohistochemistry procedure described above. Germ cell count was obtained by counting TRA98-positive germ cells in sections (30 um apart) from the entire ovary. Data were analyzed using one-way ANOVA followed by Tukey test for pair wise comparisons.Ctnnb1floxed/floxed female mice that were plugged by SF-1/cre; +/−β-Ctnnb1 male were injected daily with flutamide subcutaneously (100 mg/kg/daily) from 12.5 dpc until birth Flutamide  was dissolved in 1∶1 (vol/vol) mixture of absolute ethanol and sesame oil. Ten pregnant In situ hybridization was processed according to the standard non-radioisotopic procedure using digoxigenin-labeled RNA probes for Inhbb. Gonads with mesonephroi attached were rehydrated through a methanol gradient then wash with PTW. The rehydrated gonads were treated with proteinase K (10 mg/ml) at 37°C for 12 minutes and then post-fixed in 4% paraformaldehyde/0.1% glutaraldehyde at room temperature for 20 minutes. Samples were pre-hybridized in the hybridization buffer  at 65°C for 1 hour. Then digoxigenin-labeled Inhbb RNA probe was added into the solution and samples were rotated in an oven at 65°C overnight (12–16 hours). On the Next day, samples were washing with pre-warmed hybridization buffer followed by washing with MABTL . Samples were incubated in 20% sheep serum in MABTL blocking solution at room temperature for 2 hours followed by incubating in alkaline phosphatase-conjugated anti-digoxigenin at 4°C overnight on a shaker. On the third day, after washed in MABTL three times for 1 hour each, samples were incubated in alkaline phosphates substrate in NTMTL  for color development.Tissues were fixed overnight in 4% paraformaldehyde in PBS at 4°C and dehydrated through a methanol gradient  in PTW (0.1% Tween20 in DEPC-PBS). Samples were stored in 100% methanol at −20°C up to 6 months.


Both signaling pathways positively regulate fibroblast proliferation during this reparative process, and the effect of transforming growth factor β is partially mediated by β-catenin. Other cellular processes, such as cell motility and the induction of extracellular matrix contraction, also play important roles during wound repair. We examined the function of β-catenin and its interaction with transforming growth factor β in cell motility and the induction of collagen lattice contraction.Floating three dimensional collagen lattices seeded with cells expressing conditional null and stabilized β-catenin alleles, showed a modest negative relationship between β-catenin level and the degree of lattice contraction. Transforming growth factor β had a more dramatic effect, positively regulating lattice contraction. In contrast to the situation in the regulation of cell proliferation, this effect of transforming growth factor β was not mediated by β-catenin. Treating wild-type cells or primary human fibroblasts with dickkopf-1, which inhibits β-catenin, or lithium, which stimulates β-catenin produced similar results. Scratch wound assays and Boyden chamber motility studies using these same cells found that β-catenin positively regulated cell motility, while transforming growth factor β had little effect.This data demonstrates the complexity of the interaction of various signaling pathways in the regulation of cell behavior during wound repair. Cell motility and the induction of collagen lattice contraction are not always coupled, and are likely regulated by different intracellular mechanisms. There is unlikely to be a single signaling pathway that acts as master regulator of fibroblast behavior in wound repair. β-catenin plays dominant role regulating cell motility, while transforming growth factor β plays a dominant role regulating the induction of collagen lattice contraction. Wound healing proceeds through overlapping inflammatory, proliferative and remodeling phases. During the proliferative phase of wound healing, activated fibroblasts induce contraction of the healing wound, move across tissue defects to provide mechanical stability, and act to reorganize the extracellular matrix . These cTransforming growth factor β ligands act through Smad transcription factors to regulate gene expression. These growth factors are expressed during the initial phases of wound repair. Injection of transforming growth factor β – one into cutaneous wounds causes a larger sized wound, and absence of the transforming growth factor β regulated transcription factor, Smad3, results in early wound closure with a smaller wound size -10. TranFibroblast-like cells in the dermal compartment of the healing skin during the proliferative phase of wound healing, exhibit high levels of β-catenin protein, and activation of β-catenin mediated tcf dependent transcriptional activity ,15. Duriβ-catenin has an important role in wound healing. It also mediates the effect of transforming growth factor β in regulating proliferation during wound repair ,16,17. APrimary dermal fibroblast cell cultures were established from mice expressing conditional null or stabilized alleles of β-catenin, and wild type littermates. To activate the conditional alleles, cells were treated with an adenovirus engineered to expresses cre-recombinase. Cells fTransforming growth factor β one is known to induce contraction of three dimensional collagen lattices seeded with fibroblasts . Since βLithium will elevate β-catenin level through its regulation of GSK3β ,26, and To determine if human cells behaved the same as cells from mice, we examined human primary fibroblasts in a similar manner. Contraction was compared between cells treated with transforming growth factor β, Dkk-1, lithium, these agents in combination, or with controls. A similar pattern as found in the mouse cultures was observed. Lithium and Dkk-1 have a mild effect on lattice contraction, while transforming growth factor β has a more dramatic positive effect Fig. . Dkk-1 aThe scratch wound assay can be used to study cell migration, and approximates some of the conditions present during wound repair . Using tα-smooth muscle actin can regulate fibroblast contraction, and the expression of this gene is known to be regulated by transforming growth factor β ,31. As sDuring wound repair, fibroblasts move across tissue defects, and cause contraction of the extracellular matrix. Fibroblast motility is initiated when a cell extends a protrusion at its front which attaches to the substratum on which the cell is migrating, followed by a contraction that moves the cell body forward toward the protrusion, and finally the attachments at the cell rear release as the cell moves forward. The ability of fibroblasts to induce contraction of a collagen lattice is related to activation of fibroblast contractile machinery, the ability to transmit the contractile force to the extracellular matrix, and to the remodeling of the matrix. We found that different signaling pathways regulate these processes, suggesting that these two processes are controlled by different intracellular mechanisms.Fibroblast activation to myofibroblasts is mediated by α-smooth muscle actin, and can be induced by transforming growth factor β. This is one mechanism by which transforming growth factor β can activate the fibroblast contractile machinery ,32. We fWe found little effect of transforming growth factor β on fibroblast motility. This is in agreement with studies suggesting an inhibitory effect of transforming growth factor β on fibroblast motility ,33. HoweDuring wound repair, numerous factors contribute to wound size including the number of cells present (a function of cell proliferation and migration) and the behavior of the cells within the extracellular matrix. Activation of number of signaling pathways, such as through transforming growth factor β and β-catenin, cause a larger wound size. Our data, in concert with data from previous studies, suggests that these two signaling pathways activate different cellular processes to produce a larger wound size. Both transforming growth factor β and β-catenin positively regulate fibroblast proliferation, suggesting that this is a common cellular process in the generation of a hypertrophic wound. In contrast β-catenin has a dominant role regulates cell motility while transforming growth factor β has a dominant role regulating lattice contraction. Such data likely has important implications in therapeutic approaches to hyperplasic wound healing, as the modulation of a multiple involved signaling pathways may be required.Cutaneous wound healing is a complex process involving multiple cell types and intracellular signaling pathways. β-catenin and transforming growth factor β play important roles in this process, both of which positively regulate wound size. Here we show that transforming growth factor β plays a major regulatory role, while β-catenin plays a minor role regulating contraction of a floating collagen lattice. In contrast, we found little effect of transforming growth factor β on fibroblast motility, while β-catenin plays a significant role positively regulating fibroblast cell migration. Although β-catenin partially mediates the effect of transforming growth factor β on cell proliferation  in fibroCatnbtm2Kem mice contain β-catenin alleles with loxP sites flanking exons 1 and 6. When the segment between loxP sites is excised by exposure to cre-recombinase this effectively abolishes the ability to express β-catenin protein[Catnblox(ex3) mouse harbors a conditional β-catenin allele containing loxP sites flanking exon three. When exposed to cre-recombinase, this results in expression of a functional β-catenin protein that is missing the amino terminal phosphorylation sites and as such is a constitutively stabilized, transcriptionally active protein[Primary cell dermal fibroblast cell cultures were established from mice or from patients undergoing surgery as previously reported,36,37. In protein. The Cate protein. This re8 PFU of the virus as in our previous work. In cells from the Catnbtm2Kem mouse, this did not completely abolish β-catenin protein level, but reduced the level of to about one third of control levels, and in cells from the Catnblox(ex3) mouse, treatment doubled the β-catenin protein level. To determine if Wnt ligands are required play a role in the regulation of the lattice contraction, we treated cells with a dickkopf-1 (Dkk1) expressing adenovirus (Ad-Dkk1) as previously reported [To drive cre-recombinase expression in the murine fibroblast cells, we used an adenovirus engineered to expresses cre-recombinase (Ad-cre). Primaryreported . Dkk-1 ireported . Ad-Dkk1reported ,41. An areported ,40. Lithreported ,26. Sodireported ,43.5cells/ml of matrix, respectively. The cell-collagen mixture was aliquoted into 24 well culture dishes (0.5 ml/well) that were pre-treated with a PBS + 2% BSA solution. α-MEM, with or without 10% fetal calf serum (FCS) was added atop FPCLs in each well after polymerization. The attached FPCL were mechanically released from the sides of the culture plates. Digital images of the contracting FPCL were captured at various time points over 7 days using a conventional flatbed Cannon scanner. Average collagen lattice diameter was then measured using imaging software . Collagen lattices were prepared by mixing cells with a neutralized solution of collagen type I . Final collagen and cell concentrations for the FPCL were 2.0 mg/ml and 3 × 10rd et al ,45). At rd et al . Each in4 cells were seeded into 35-mm plastic tissue culture plates. Confluent monolayers were obtained after three days, afterwhich the cells were incubated for 12 hours in serum free media. A "scratch" in the middle of the cell monolayer was produced using a 1 mm wide cell scraper. Cells were observed immediately after the generation of the scratch to ensure a uniform 1 mm wide "scratch" region. They were then observed again 24 hours later to measure the average distance between the cells on each side of the scratch as previously reported [1.0 × 10reported . A 6-mm Real time PCR was used to determine differences in alpha smooth muscle actin using previously reported techniques. Cells dRP carried out the collagen contraction assays, SAN, JA, and LS carried out the scrtach and motility assays. BAA concieved conceived of the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


CTNNB1 exon 3, we detected a stabilizing mutation (S37A) in 3 out of 20 analyzed adenomas. The aim of the present study was to determine the frequency and zygosity of mutations in CTNNB1 exon 3, and β-catenin accumulation in a large series of parathyroid adenomas of Swedish patients.Aberrant accumulation of β-catenin plays an important role in a variety of human neoplasms. We recently reported accumulation of β-catenin in parathyroid adenomas from patients with primary hyperparathyroidism (pHPT). In The mutation S37A (TCT > GCT) was detected by direct DNA sequencing of PCR fragments in 6 out of 104 sporadic parathyroid adenomas (5.8%). Taking our previous study into account, a total of 9 out of 124 (7.3%) adenomas displayed the same mutation. The mutations were homozygous by DNA sequencing, restriction enzyme cleavage, and gene copy number determination using the GeneChip 500 K Mapping Array Set. All tumors analyzed by immunohistochemistry, including those with mutation, displayed aberrant β-catenin accumulation. Western blotting revealed a slightly higher expression level of β-catenin and nonphosphorylated active β-catenin in tumors with mutation compared to those without. Presence of the mutation was not related to distinct clinical characteristics.Aberrant accumulation of β-catenin is very common in parathyroid tumors, and is caused by stabilizing homozygous mutation in 7.3% of Swedish pHPT patients. CCND1 oncogene expression or inactivation of the MEN1 tumor suppressor gene contributes to deregulated growth control in a fraction of sporadic parathyroid adenomas [Parathyroid disease with hypersecretion of parathyroid hormone and generally also hypercalcemia occurs in primary hyperparathyroidism (pHPT), due to growth regulatory disturbance in one or several parathyroid glands. Activation of adenomas .Activation of the Wnt/β-catenin signaling pathway by aberrant accumulation of stabilized β-catenin is involved in the development of many neoplasms. β-catenin accumulation is typically caused by mutations in components of the signaling pathway, such as APC, Axin, β-Trcp, and WTX, or results from secondary events. In addition, protein stabilizing mutations in the glycogen synthase kinase 3β phosphorylation sites of β-catenin  occur with varying frequency in several neoplasms .CTNNB1 exon 3 [CTNNB1 have not been detected in parathyroid adenomas of patients from Japan and the United States [CTNNB1, and β-catenin expression status in a large series of parathyroid adenomas of Swedish patients.We recently reported activation of the Wnt/β-catenin signaling pathway by aberrant accumulation of β-catenin in parathyroid adenomas from patients with pHPT . The acc1 exon 3 ,11. Stabd States ,13. HereSporadic parathyroid adenomas (n = 104) were acquired from 104 Swedish patients with pHPT diagnosed and operated on in the clinical routine at the Uppsala University Hospital. Normal parathyroid tissue was obtained as normal gland biopsies in patients subjected to parathyroidectomy. Tissues were intraoperatively snap frozen. Informed consent and approval of institutional ethical committee were obtained.CTNNB1. PCR forward primer: 5'-TGA TGG AGT TGG ACA TGG CC; reverse: 5'-CTC ATA CAG GAC TTG GGA GG. The complementary strand was also sequenced for fragments with mutation. The PCR fragments were sequenced directly on the 3130xl Genetic Analyzer using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction kit .DNA from parathyroid tumors was prepared by standard procedures including proteinase K treatment and phenol extraction. Blood DNA was prepared using the Wizard Genomic DNA Purification Kit . DNA was PCR amplified with primers for exon 3 of CTNNB1 exon 3 PCR fragments were purified using the GFX PCR DNA and Gel Band Purification Kit  and cleaved with Xma I or Nla III according to instructions by the manufacturer . Products were analyzed by agarose gel electrophoresis.Tumor (S37A) and blood DNA from 4 patients were extracted as described above. DNA was marked with fluorescence dye and hybridized to the Affymetrix GeneChip Mapping 500 K Set Arrays 250K_Nsp_SNP and 250K_Sty_SNP according to the manufacturer's instructions, and analysed by GeneChip Genotyping Analysis Software (GTYPE) using Chromosome Copy Number Analysis Tool (CNAT) . Informative SNPs used in the gene copy number determination are shown in Table Frozen tissue sections were stained as described  using ant test, z test, and χ2 test were used. The data were calculated with Statistica 6 . Values are presented as arithmetrical mean ± SEM.Unpaired CTNNB1 stabilizing mutation S37A (TCT > GCT) in 6 out of the 104 (5.8%) analyzed parathyroid adenomas . Gene copy number analysis was done by comparison of informative single-nucleotide polymorphisms (SNPs). Five informative SNPs in Previously, we reported aberrant accumulation of β-catenin in all n = 37) analyzed parathyroid adenomas . Of the 7 analyzeCTNNB1 stabilizing homozygous mutation S37A. None of these 9 tumors expressed the internally truncated LRP5 receptor [We have found that a total of 9 out of 124 (7.3%) randomly selected parathyroid adenomas displayed the receptor   to mechanisms of parathyroid disease. The CTNNB1 mutation frequency vary considerably also in colorectal cancer (1–60%) and melanomas (0.02–27%), apparently not related to geographical origin [DNA sequence analysis of 24 parathyroid adenomas from Japanese patients revealed no 2 tumors . In anot2 tumors . Furthers report . Since wl origin ,17,22-30CTNNB1-stabilizing mutations were found in 5.8% of the tumors, or in 7.3% when taking our previous study [By analyzing a large series of tumors from Swedish patients, this study further emphasizes β-catenin accumulation as the most common aberration in parathyroid tumors of primary origin. us study  into accus study  in partiThe authors declare that they have no competing interests.PB participated in the design of the study, performed experiments, analyzed data and performed the statistical analysis, DL analyzed the data, GÅ helped to draft the manuscript, GW conceived of the study, participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.


We thus sought to determine whether gastrin might regulate β-catenin expression in colorectal tumour cells. Amidated gastrin-17 (G-17), one of the major circulating forms of gastrin, not only enhanced β-catenin protein expression, but also one of its target genes, cyclin D1. Furthermore, activation of β-catenin-dependent transcription by gastrin was confirmed by an increase in LEF-1 reporter activity, as well as enhanced cyclin D1 promoter activity. Finally, G-17 prolonged the τ1/2 of β-catenin protein, demonstrating that gastrin appears to exert its mitogenic effects on colorectal tumour cells, at least in part, by stabilising β-catenin.As gastrin may play a role in the pathophysiology of gastrointestinal (GI) malignancies, the elucidation of the mechanisms governing gastrin-induced proliferation has recently gained considerable interest. Several studies have reported that a large percentage of colorectal tumours overexpress or stabilise the  Gastrin was originally described as a gastrointestinal (GI) regulatory peptide whose principal function was to stimulate postprandial gastric acid secretion. However, in addition to its recognised role in the physiological regulation of acid secretion, another biological property attributed to gastrin is its trophic effects. A prospective study by β-catenin oncogene in the pathogenesis of CRC  in the N-terminus , a serine/threonine kinase that is overexpressed in many malignancies, has been shown to phosphorylate β-catenin in the midportion of the protein and enhance its stability (Overwhelming evidence derived from studies involving primary colon tumours of both hereditary and sporadic origin has implicated aberrations of the adenomatous polyposis coli (APC) tumour suppressor gene and tability .β-catenin escapes its negative regulatory mechanisms, it translocates into the nucleus and functions as a critical transcriptional coactivator of the T-cell factor/lymphocyte enhancer binding factor (TCF/LEF), which activates oncogenes, such as c-myc , in which the peptide caused a significant incorporation of [3H]thymidine at 24 and 48 h  for 2–4 h, and 1 μM of the gastrin-specific receptor antagonist L365,260  was used in conjunction with G-17 in the indicated experiments. Cycloheximide, a de novo protein synthesis inhibitor, was used at a final concentration of 10 μg ml−1, either alone or in combination with 20 or 50 nM G-17 for the indicated times .We utilised MC-26 mouse CRC cells, which were maintained in Dulbecco's modified Eagle's Medium , supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin/streptomycin. Amidated G-17  was added to the culture medium (20–100 nμg) was loaded onto a formaldehyde-containing agarose gel and transferred via capillary action overnight onto a Hybond-N nylon membrane  in 10 × SSC buffer. Transferred membrane was crosslinked and prehybridised prior to the addition of the labelled probe. Approximately 1 kb fragment of β-catenin cDNA and 3.2 kb fragment of actin cDNA were excised and used as probes. Both of the probes were labelled with [32P]dCTP for 30 min, purified with Quick Spin sephadex G-25 columns , boiled, and incubated with prehybridised membrane overnight at 65°C. Labelled membranes were washed four times, twice in 2 × SSC/0.1% SDS and twice in 0.2 × SSC/0.1% SDS, before exposing to a film. The membrane was briefly stripped with boiling 0.1% SDS and washed 3 × with 2 × SSC before addition of another probe.Total RNA was extracted using the Qiagen RNeasy kit  following the manufacturer's instructions. Each RNA sample  and cyclin D1 . Ponceau S  staining and immunoblots with either a monoclonal β-actin antibody (Sigma) for whole-cell lysates or polyclonal Sp1 antibody  for nuclear extracts were used to confirm equal loading of Western blot membranes.Western blotting analyses were performed, as previously described , using a5) were plated 1 day prior to transfection. For transient transfection experiments, subconfluent cells were incubated with DNA and FUGENE-6 liposome reagent  according to the manufacturer's instructions. Both LEF-1 and cyclin D1  in a luminometer for 10 s. Subsequently, in the same tube, 50 μl renilla substrate (Promega) were added and measured. Samples were assayed in duplicate, and the luciferase counts were normalised to renilla measurements.MC-26 cells (1–2 × 10μg) were assayed for CK2 kinase activity, as previously described  were also measured and subtracted from the final radioactive counts.Equal amounts of protein extracted from MC-26 cells  contains eight repeats of the LEF binding site that is activated only in the presence of an exogenous LEF-1 construct. The level of LEF-1-dependent transcription is also dependent on nuclear β-catenin levels, as β-catenin is a known coactivator for TCF/LEF transcription factors. As we speculated that an increase in β-catenin protein by G-17 might be functionally important for the transcriptional activation of its target genes, LEF-1-dependent reporter assays were performed in the absence and presence of G-17. We observed that 20 and 50 nM G-17 induced a concentration-dependent increase in LEF-1-dependent transcriptional activity (P⩽0.005)  when compared to either the empty or minimal promoter (−66) (P⩽0.01).To examine whether the increase in nuclear P⩽0.005) . In addincreased . Specifier (−66) , P⩽0.01.β-catenin transcripts but increased β-catenin protein levels, protein stability of β-catenin was next examined. To determine this, MC-26 cells were incubated with cycloheximide, a de novo protein synthesis inhibitor, either in the absence or presence of 20 and 50 nM G-17. Because β-catenin is normally degraded by proteasomes, the addition of cycloheximide would enable the pool of translated cytoplasmic protein to be degraded at its natural rate. Total protein was extracted at 0, 3, and 6 h, and β-catenin protein levels were measured by Western analysis. In the presence of cycloheximide alone, nearly 50% of β-catenin was degraded by 3 h . In contrast, 20 and 50 nM G-17 caused a marked increase in endogenous CK2 kinase activity  mice produced fewer polyps than APC (min−/+) mice overexpressing gastrin. Furthermore, these investigators showed that β-catenin enhanced gastrin promoter activity, thus identifying gastrin as one of its numerous downstream targets. However, the possibility of a positive feedback relationship between gastrin and β-catenin expression has not been examined previously.Both gastrin and various components of the s of CRC . Howeverβ-catenin protein. We consistently observed that gastrin increases β-catenin protein levels. However, despite our attempts to maintain consistency, such as plating equal amounts of cells 1 day prior to each individual experiment, we nevertheless did observe some variability in the basal expression (untreated) of β-catenin during the performance of different experiments. This variability may be due in part to the role of β-catenin in cell–cell adhesion, which, depending on cell density, may contribute to the variability in basal β-catenin expression.Utilising transplantable mouse CRC cells (MC-26) that express functional gastrin receptors, we have previously demonstrated the trophic properties of gastrin  involving β-catenin expression by gastrin was also associated with the enhancement of the critical cell cycle regulator, cyclin D1. Consistent with our current findings, we have previously reported that gastrin enhanced cyclin D1 protein and cyclin D1 promoter activity in the human gastric adenocarcinoma cell line AGS-B  (myc levels in this study, it is certainly possible that gastrin may involve not only c-myc and cyclin D1, but also multiple β-catenin target genes and pathways in exerting its growth potential, whether directly or indirectly. Another possibility is simply the fact that every immortal cell line possesses slightly different characteristics that produce disparate results. For example, unlike AGS-B cells (β-catenin and CREB pathways (The upregulation of β-catenin protein by prolonging its half-life. Furthermore, these studies support our hypothesis that a positive feedback mechanism exists between gastrin and β-catenin. Although further studies will be required to elucidate fully the mechanisms governing gastrin-induced cellular proliferation, our results suggest that through aberrant overexpression of β-catenin, malignant cells appear to amplify various signals via positive feedback between molecules as a means for potentiating tumorigenesis and proliferation.In conclusion, the results of the present studies demonstrate for the first time that gastrin enhances


Presenilin1 (PS1) is a component of the γ-secretase complex mutated in cases of Familial Alzheimer's disease (FAD). PS1 is synthesized as a 50 kDa peptide subsequently processed to two 29 and 20 kDa subunits that remain associated. Processing of PS1 is inhibited by several mutations detected in FAD patients. PS1 acts as negative modulator of β-catenin·Tcf-4 transcriptional activity. In this article we show that in murine embryonic fibroblasts (MEFs) the mechanisms of action of the processed and non-processed forms of PS1 on β-catenin·Tcf-4 transcription are different. Whereas non-processed PS1 inhibits β-catenin·Tcf-4 activity through a mechanism independent of γ-secretase and associated with the interaction of this protein with plakoglobin and Tcf-4, the effect of processed PS1 is prevented by γ-secretase inhibitors, and requires its interaction with E- or N-cadherin and the generation of cytosolic terminal fragments of these two cadherins, which in turn destabilize the β-catenin transcriptional cofactor CBP. Accordingly, the two forms of PS1 interact differently with E-cadherin or β-catenin and plakoglobin: whereas processed PS1 binds E-cadherin with high affinity and β-catenin or plakoglobin weakly, the non-processed form behaves inversely. Moreover, contrarily to processed PS1, that decreases the levels of c-fos RNA, non-processed PS1 inhibits the expression c-myc, a known target of β-catenin·Tcf-4, and does not block the activity of other transcriptional factors requiring CBP. These results indicate that prevention of PS1 processing in FAD affects the mechanism of repression of the transcriptional activity dependent on β-catenin. Presenilin 1 (PS1) encodes a ubiquitously expressed, eight-transmembrane protein involved in most cases of early-onset Familial Alzheimer's disease (FAD) PS1 assembles with nicastrin, aph-1 and pen-2 to form the large γ-secretase complex, responsible for the cleavage of several type-I transmembrane proteins, including the β-amyloid precursor protein (APP), Notch, CD44, ErbB4, E-cadherin and N-cadherin Drosophila presenilin (DPS) works as a negative modifier of wingless/Wnt in genetic screening In recent years, PS1 has been identified as an important negative regulator of β-catenin signaling. MycA/His. The presence of the myc-tag at the C-terminal end of wild-type PS1 impedes the endoproteolytic processing of the protein. Full-length human CBP was cloned into the BamHI-NotI site of pcDNA3.1-HA. Full-length cDNA of murine E-cadherin was released from pBATEM2 plasmid by digestion with BglII-HindIII and cloned into BglII-HindIII-linearized pECFP-N1 vector. The DNA fragments corresponding to the cytosolic domain of E-cadherin  or N-cadherin  were amplified from murine RNA by RT-PCR using oligonucleotides corresponding to nucleotide sequences 2194–2219/2623–2649 and 2235–2253/2699–2717, respectively. The 0.5 kilobase amplification fragments were inserted in the BamHI-EcoRI site of a pGEX-6P3 plasmid for E-cadherin, or in the BamHI-XhoI site of a pGEX-6P1 plasmid for N-cadherin. To generate the eukaryotic expression plasmid, the cytosolic cDNA fragments of E- and N-cadherin were released from the pGEX-6P plasmid by digestion with BamHI-EcoRI for E-cadherin and with BamHI-XhoI for N-cadherin and cloned into BglII-EcoRI-linearized pEGFP-C1 or BamHI-XhoI-linearized pcDNA3.1, respectively. The preparation of the plasmids codifying for p120-catenin, Tcf-4 and β-catenin has been reported elsewhere Full-length human PS1 and the point mutants M146L H163R, C410Y and deleted in exon 9 (Δe9), kindly provided by Dr. C.A. Saura  were cloned in pcDNA3.1 plasmid. Alternatively, full-length PS1 was inserted in the BamHI-EcoRV sites of pcDNA3.1E. coli and purified by affinity chromatography on glutathione-Sepharose 2, 0.5 mM EGTA, 1 mM DTT, 0.5 mM EDTA, 28 mM α-glycerolphosphate pH 7.0, 0.1 mM ATP), using 22 pmols of GST-E-cadh-CTF2, GST-N-cadh-CTF2 or GST as a control, and 300 milliunits of protein kinase CKIα (Sigma). Reactions were performed for 40 min at 30°C. When required for the pull-down assays, GST was removed by cleaving with Pre-Scission protease (Amersham Biosciences). Pull-down assays were performed by using purified recombinant proteins fused to GST and cell extracts from MEF PS (+/+) cells or deficient for PS1 and PS2 (MEFs PS (−/−)), as described before GST-fusion proteins corresponding to the indicated forms of p120-catenin and the cytoplasmic domains of E-cadherin (E-cadh-CTF2) or N-cadherin (N-cadh-CTF2) were expressed in The cell lines SW-480 and murine embryonic fibroblast wild type (MEF PS (+/+)) or deficient for PS1 and PS2 (MEF PS (−/−)) were used in this study. MEF PS (−/−) were generously provided by Dr. B. De Strooper Cytosolic fractions were prepared by homogenizing cells in RIPA buffer , supplemented with protease inhibitors and phosphatase inhibitors. Cell homogenates were left on ice for 15 min and centrifuged at 500 xg for 10 min at 4°C to obtain the cytosolic fractions. Pellets were resuspended in the same volume of RIPA buffer supplemented with protease and phosphatase inhibitors. After passing cells 20 times by a insulin syringe, extracts were left on ice for 15 min and centrifuged at 20.000 xg for 10 min at 4°C. Supernatants constituted the nuclear fraction.Proteins were immunoprecipitated from cell extracts (300 to 600 µg) by using 4 µg/ml of the appropriate antibody for 16 h at 4°C. Precipitated material was removed by centrifugation at 12,000 xg and the resulting supernatant was incubated for 90 min with 30 µl of protein A-agarose (Sigma). Immunoprecipitates were washed three times with lysis buffer and bound proteins directly eluted with electrophoresis sample buffer and analyzed by Western blotting. When appropriate, mouse TrueBlot reagent (BD Biosciences) was used as a secondary antibody in order to eliminate interferences in the autoradiogram.Renilla luciferase gene under the control of a constitutive thymidine kinase promoter (Promega) was used as a control. Assays were always performed in triplicate; the average of the results of three-four independent transfections±S.D. is given.Cells were transfected as above with the different promoters. TOP-Flash plasmid, a synthetic promoter sensitive to the activity of the β-catenin·Tcf-4 complex, that contains three copies of the Tcf-4 binding site upstream of a firefly luciferase reporter gene P values of <0.05 were considered to be significant in each case.Statistical analyses were carried out using SPSS software version 14. The results of the means percentage were compared by ANOVA. Myc/His-PS1 (npPS1) or empty vector and 1.5 µg pBABE-puro hrGFP. After 48 h, transfected cells were selected with medium containing 2.5 µg/ml Puromycin for additional 48 hours. Total cell protein extracts were prepared as indicated above; RNAs were obtained as previously reported MEF PS (−/−) cells were cotransfected with 10.5 µg of either pcDNA3.1-PS1 (pPS1), pcDNA3.1-We analyzed the effect of two different forms of PS1 on the activity of TOP reporter plasmid, a widely used system to determine β-catenin·Tcf-4 transcriptional activity. An untagged form of PS1 was partially processed when transfected to epithelial intestinal SW-480 cells . HoweverSimilar experiments were performed in murine embryo fibroblasts (MEFs) obtained from PS1 and PS2 knock-out animals, transfecting the two PS1 forms to levels similar to those detected in control MEFs (+/+) . The tagThe effect of all these PS1 forms on the TOP activity was also determined. Although both the processed and non-processed forms of PS1 inhibited this activity, the non-processed proteins  did it to a higher extent . This diTherefore, these results indicate that PS1 presents γ-secretase-dependent and -independent inhibitory effects on β-catenin·Tcf-4-dependent transcription that are related to its own processing.Inhibition of β-catenin·Tcf-4 transcriptional activity by npPS1 is related to its ability to interact with plakoglobin We further investigated the mechanism involved in the repression of β-catenin·Tcf-4 transcriptional activity by pPS1 in MEFs. Deficiency of PS1 and PS2 caused increased levels of E-cadherin, N-cadherin, plakoglobin and, to a lesser extent, β-catenin . The expIt has been proposed that PS1 promotes the degradation of β-catenin and, as a consequence of lower availability of this protein, inhibits β-catenin translocation to the nucleus and its transcriptional activity PS1-deficient MEFs showed higher levels of E- and N-cadherin than wild-type MEFs. For both cadherins, full-length proteins and C-terminal fragments, CTF1 (at 37 kDa for E-cadherin and 39 kDa for N-cadherin) were detected at higher levels in MEFs (−/−) than in MEFs (+/+) . AdditioThe destabilization of E-cadherin by PS1 was also observed when both proteins were ectopically expressed in MEFs (−/−). As shown in The direct interaction of E-cadherin with the processed and non-processed PS1 forms was also determined by pull-down assays. As observed in It has been reported that PS1 associates with E-cadherin at the p120-catenin binding site Next, we checked the influence of p120-catenin on the effect of PS1 on β-catenin·Tcf-4 transcriptional activity. Addition of p120-catenin prevented the repression caused by pPS1 of TOP promoter activity without affecting that caused by npPS1 , suggestSubcellular location of CTF2, determined transfecting this protein tagged with GFP, indicated that this protein was detected exclusively in the cytosol (data not shown). This result, and previous observations shown in N-cadherin CTF2 is involved in the down-regulation of CBP Supplementation with exogenous CBP prevented the repression of β-catenin·Tcf-4-dependent transcription caused by expression of pPS1 in MEFs (−/−)  and stimin vivo. The interaction was also detected by co-immunoprecipitation using cytosolic fractions and a previous treatment with proteasome inhibitors such as MG132, in order to prevent the degradation of CBP. As shown in Down-regulation of CBP levels by N-cadherin-CTF2 has been reported to be dependent on their interaction We also analyzed several different properties of the transcriptional repression by processed and non-processed PS1. Since npPS1 affects β-catenin·Tcf-4 transcriptional activity by sequestering Tcf-4 in non-functional complexes, inhibition by this PS1 form is prevented by Tcf-4 over-expression Since pPS1 decreases the cell levels of CBP, we expected that it not only affects the activity of β-catenin·Tcf-4 complex but also that of other transcriptional factors dependent on this cofactor, such as CREB (cyclic AMP response-element binding protein). Binding of CBP is required for stimulation by CREB of the expression of genes containing CREs (cyclic AMP responsive elements) in their promoters. Therefore, we checked the effects of both PS1 forms on the activity of a CRE luciferase reporter plasmid in cells stimulated with forskolin. Whereas npPS1 did not affect the activity of this promoter, pPS1 repressed it by 50% . As expeCBP also modulates the activity of NF-κB Finally, we analyzed the effect of the two PS1 forms on gene expression in stable MEFs transfectants. Both forms differently affected cell proliferation: whereas MEF(−/−) cells expressing npPS1 proliferated at a rate 38% of control cells, those expressing pPS1 did it at a 60% . Since cThe effect of both PS1 forms on c-Fos RNA was also examined, since expression of this gene has been reported to be sensitive to CREB, pPS1 and the γ-secretase inhibitor L-685,458 Therefore these results indicate that processed and non-processed PS1 act differently on gene transcription.In recent years, a growing amount of evidence has indicated that PS1 controls the transcriptional activity of the β-catenin·Tcf-4 complex. Different mechanisms have been put forward, involving the interaction of PS1 with N- and E-cadherin and β-catenin and the activity of the γ-secretase complex When processed, PS1 affects β-catenin·Tcf-4 transcription through a completely different mechanism, in this case dependent on its γ-secretase activity. Unlike npPS1, pPS1 has a detectable association with E or N-cadherin and facilitates the generation of a C-terminal fragment of both proteins consisting of the cytosolic domain (CTF2). It is not yet known whether the interaction of PS1 with cadherins is direct or mediated by other membrane components since the binding experiments have not been performed using pure proteins. Therefore, the lack of interaction of npPS1 with cadherins may be due to its inability to associate with the factor mediating this interaction, maybe because of a hampered localization of npPS1 in the cytoplasmic membrane. This lack of interaction with cadherins might be a more general property of mutant PS1 forms, since many of these mutants show defects in their cell trafficking and, presumably, are not located in the cell membrane It should be noted that pPS1 affects the stability of E- and N-cadherin, although γ-secretase does not participate in the processing of the full-length protein, but of the CTF1 fragment. Since pPS1 binding competes with that of p120-catenin, it is possible that pPS1 prevents the stabilization caused by this catenin on E-cadherin The effect of pPS1 on E-cadherin stability is also demonstrated by the remarkable levels of expression of this protein in MEFs deficient for PS1. Actually these cells present a more epithelioid phenotype than wild type MEFs (data not shown). This unexpected result suggests that in these fibroblasts E-cadherin expression is not exclusively modulated at the transcriptional level, as it is generally considered Unlike what happens with E-cadherin, npPS1 associates with β-catenin or plakoglobin much better than pPS1. It has been reported that binding of PS1 to β-catenin would be direct through the 298–380 hydrophilic loop Our results totally agree with previous reports indicating that inhibition β-catenin transcriptional activity by pPS1 is dependent on γ-secretase-dependent generation of N-cadherin CTF2, a fragment that induce the destabilization of the transcriptional coactivator CBP


In culture pluripotency is maintained by leukemia inhibitory factor (LIF) stimulation of two separate signaling axes: Stat3/Klf4/Sox2 and PI3K/Tbx3/Nanog, which converge in the regulation of Oct4 expression. However, LIF signaling is not required in vivo for self-renewal, thus alternate signaling axes likely mediate these pathways. Additional factors that promote pluripotency gene expression have been identified, including the direct regulation of Oct4 by liver receptor homolog-1 (Lrh-1) and β-catenin regulation of Nanog. Here, we present genetic, molecular, and pharmacological studies identifying a signaling axis in which β-catenin promotes pluripotency gene expression in an Lrh-1-dependent manner. Furthermore, Lrh-1 was identified as a novel β-catenin target gene, and Lrh-1 regulation is required for maintaining proper levels of Oct4, Nanog, and Tbx3. Elucidation of this pathway provides an alternate mechanism by which the primary pluripotency axis may be regulated in vivo and may pave the way for small molecule applications to manipulate pluripotency or improve the efficiency of somatic cell reprogramming. S The Oct4/Sox2/Nanog pluripotency axis has consistently been reiterated as the central pathway mediating ESC pluripotency and self-renewal and more recently in reprogramming somatic cells into induced pluripotent stem cells in both the mouse and human model system –5. Stand-/- ESC maintain expression of Oct4. Thus, Lrh-1 is not essential for maintaining ESC self-renewal, but rather is principal in regulating Oct4 expression during early differentiation [Previously we reported that the orphan nuclear receptor, Lrh-1, is essential for maintaining Oct4 expression at the epiblast stage of embryonic development, and that genetic ablation causes embryonic lethality around 6.0–7.0 days postcoitum (dpc) . This wantiation .Lrh-1 null mice, β-catenin null mice fail to gastrulate and die at approximately E6.0 [Others have implicated Lrh-1 as a central factor governing the self-renewal of intestinal crypt cells by forming a complex with β-catenin . The ubiely E6.0 . β-catenely E6.0 ,21. β-caely E6.0 –24, howeely E6.0 ,26. Amonely E6.0 . In suppely E6.0 .catenin-/- ESC. Although exhibiting defects in differentiation, the β-catenin-/- ESCs maintain expression of pluripotency genes, albeit at decreased levels, and serve as a stringent control for molecular studies. Among those, we observe that Lrh-1 expression is induced on stimulation of Wnt signaling using molecular and pharmacological methods, and induction occurs in a β-catenin-dependent manner. Furthermore, Wnt induction of pluripotency factors is both β-catenin and Lrh-1 dependent. We report that β-catenin regulates Lrh-1 directly via conserved lymphoid enhancer factor (LEF)/T-cell factor (TCF) sites in the full-length and embryonic stem (ES) specific promoters of Lrh-1 [Tbx3, Nanog, and Oct4. Finally, ectopic expression of Lrh-1 in β-catenin-/- ESC is sufficient to restore Oct4 and Nanog expression to wt levels.To investigate definitively the role of canonical Wnt signaling in the regulation of pluripotency, we generated β-of Lrh-1 , allowinBy implementing a genetic approach in the ESC model, we have revealed a secondary pluripotency axis driven by canonical Wnt regulation of Lrh-1. Elucidation of this pathway extends our bourgeoning understanding of the molecular signature of pluripotency, and may prove applicable to reprogramming and other ESC applications.cateninflox/flox male mice  were crossed with transgenic females expressing Cre-recombinase driven by the Zona Pelucida protein-3 (ZP3) promoter (Jackson Laboratories) triggering an early recombination event [catenin+/flox, ZP3-Cre/+ females were derived and backcrossed to wt males to yield β-catenin-/+ offspring. Resultant β-catenin-/+ offspring were isolated and maintained in a colony . Genotyping was performed using the following specific primers for the β-catenin locus: RM41: 5′-AAGGTGGAGTGATGAAA- GTTGTT-3′, RM42: 5′-CACCATGTCCTCTGTCTATTC-3′, and RM43: 5′-TACA CTATTGAATCACAGGGACTT-3′ [DNA was extracted from ES cell lines and mouse tails after overnight digestion in lysis buffer , followed by phenol/chloroform/isoamyl alchohol extraction , under standard conditions described previously [all-trans-retinoic acid . Embryoid body differentiation was carried out by forming the aggregates in low-attachment plates  or by hanging drop (500 cells/20 micro l drop) for 3 days prior to transferring the embryoid bodies to gelatinized plates. ESC lines were treated with 2 micro M BIO  reconstituted in dimethyl sulfoxide (DMSO) or 100 ng/ml mWnt3a . Recombinant Dickkopf-1  was added at increasing concentrations from 1 ng/ml to 250 ng/ml. CHIR99021  was added at an increasing concentration . All treatments were performed in the absence of LIF.ES cell lines were maintained on plates treated with 0.1% gelatin  under the following conditions: 125,000 ESCs were transfected in suspension with 100 ng of reporter, 2 ng of Renilla luciferase expression vector , using 2.5 micro l Lipofectamine 2000 (Invitrogen), and plated in single well of a 12-well plate. Each transfection was assayed in triplicate. Promoter activity was measured using a Berthold Centro LB960 dual-luciferase luminometer and recorded in relative light units after normalizing to Renilla luciferase.The Lrh-1 promoter regions were generously provided by You-Hua Xie  and wereAntibody information and conditions are listed in (Supporting Information http://www.qiagen.com) on a Step 1 Plus Real Time PCR System . Target gene expression was normalized to β-actin expression in all experiments. For gene-specific primers see Supporting Information Total RNA was extracted from ES cells using Trizol reagent (Invitrogen). cDNA was generated using the Super Script III First Strand Synthesis Kit (Invitrogen) with Oligo dT primers following the manufacturer's protocol. Quantitative expression of endogenous genes was carried out using QuantiFast SYBR Green PCR  following the manufacturers protocol. Lysate protein concentration was determined postshearing and lysates diluted to 0.25 micro g/micro l. For antibody information and immunoprecipitation conditions see (Supporting Information Chromatin immunoprecipitation (ChIP) assays were performed following a protocol modified from previous reports ,33. Undiwt and Lrh-1-/- ESC after injection of approximately 106 cells into the hind-quarters of nude SCID mice. Histological analysis was carried out on teratoma sections after standard H&E staining.Teratomas were generated from pCMV-myc expression vector (Clontech) using PCR primers: forward 5′-TCGAATTCTCATGCTGCCCA AAG TGGAG-3′ and reverse 5′-TAGTCGACTTAGGCTCTT TTGGCATGCAGCA-3′ [pEF1α-ORF-IRES-neo vector described in with PCR primers: forward 5′-TCGCTAGCATGGCA TCAA TGCAGAAGCT-3′, and reverse 5′- TCGCGGCCGCTTAGG CTCTTTTGGCATGCA-3′ [pEF1α-mycLrh1(ES)-IRES-neo vector was targeted into the β-catenin-/- ESCs via electroporation . Heterologous recombinants were screened by selection with G418 for approximately 10 days, and clonal resistant ES colonies were isolated and expanded in culture.The previously described embryonic isoform of Lrh-1 was cloned into a CAGCA-3′ . Myc-tagATGCA-3′ . The pEFt-tests where appropriate.Statistical significance was determined by performing two-tailed catenin-/- ESC were derived by blastocyst outgrowth following a germline Cre-lox recombination event yielding heterozygous mice [catenin-/- ESC express these pluripotency factors, they are not pluripotent insofar as they exhibit pronounced defects in endoderm and mesoderm induction on embryoid body differentiation  isoform driven by an alternate promoter downstream of the full-length (FL) promoter [wt and β-catenin-/- ESC to compare activity [Lrh-1 FL promoter exhibits basal activity in both cell lines, the 1.4 kb ES promoter revealed significantly higher activity in wt cells compared with the mutant cell line, which was suppressed on differentiation with RA, and induced in a β-catenin-dependent manner on BIO treatment -1, Ts(ES)-1, and Ts(ES)-2 became highly enriched after treatment with BIO -1, Ts(FL)-2, Ts(ES)-1, and Ts(ES)-2 Fig. A. We theBIO Fig. B. β-catereatment , whereasger Fig. B.Lrh-1 reporters described in Figure Tcf3-/- ESC with an increasing concentration of Tcf3 expression vector [Lrh-1 FL promoter exhibited basal activity, however, the 1.4-kb ES-specific promoter yielded high promoter activity that was repressed by Tcf3 in a concentration-dependent manner -1 and Ts(ES)-2 in the IP assay ,46. ImpoLrh-1-/- ESC to recapitulate the defect in pluripotency gene expression observed in the β-catenin-/- ESC [Lrh-1-/- ESC were differentiated as embryoid bodies or by RA-mediated differentiation under the same conditions reported in Figure catenin-/- ESC, the Lrh-1-/- ESC express significantly decreased levels of Oct4 and Nanog, while expressing similar levels of β-catenin catenin, expressed Lrh-1 at levels fourfold higher than that observed in undifferentiated wt cells  that converge in the regulation of Oct4, thereby establishing the Oct4/Sox2/Nanog autoregulatory loop ,6. Intricatenin-/- and Lrh-1-/- ESC and determining the requirement of these factors in promoting pluripotency after stimulating Wnt signaling using various molecular and pharmacological methods -mediated repression of Oct4 ,48. Thisods Fig. E. We havesources .Previous reports have identified PI3K as a component of the LIF signaling cascade that functions independent of Stat3 . Its rolTcf3-/- ESC exhibit increased and prolonged expression of Nanog and Lrh-1 during ESC differentiation [Tcf3 is reported to play an unconventional role in mediating the effects of the primary pluripotency axis by forming a complex with Oct4, Sox2, and Nanog ,46. Surpntiation . This phRecent studies have described a “ground state” to ESC pluripotency, concluding that ESC can self-renew independent of growth factors and cytokines when differentiation cues are absent, thus, ESC have an innate program directing self-renewal . WhetherIt is also of interest to see to what degree the β-catenin/Lrh-1 pathway can influence the emerging field of somatic cell reprogramming. iPSCs hold a great deal of promise for future therapeutic applications, however, caveats exist, such as the efficiency of reprogramming and their safety in application. Recently, another group identified Lrh-1 as being sufficient to replace Oct4 during viral-mediated reprogramming . Others The intricate nature of the Wnt signaling pathway makes it amenable to small molecule manipulation. Many known modulators of Wnt signaling have been characterized, and may hold potential not just in promoting somatic cell reprogramming, but perhaps in directing differentiation into specific lineages as well . AdditioHere, we present evidence that canonical Wnt/β-catenin pathway regulates Lrh-1 during ESC self-renewal thereby comprising a secondary pluripotency axis in the promotion of Oct4, Nanog, and Tbx3 gene expression. We base this finding on the incorporation of genetic, pharmacological, and molecular studies that are described herein. Furthermore, Lrh-1 was identified as novel target gene of both β-catenin and Tcf3, indicating they likely partner in regulating Lrh-1 expression in the ESC. We believe this pathway to be significant as it provides an alternate mechanism to LIF-mediated pluripotency, which is known to be dispensable in vivo ,9, and mThe authors indicate no potential conflicts of interest.


Aberrant activation of the canonical Wnt/β-catenin pathway occurs in almost all colorectal cancers and contributes to their growth, invasion and survival. Phopholipase D (PLD) has been implicated in progression of colorectal carcinoma However, an understanding of the targets and regulation of this important pathway remains incomplete and besides, relationship between Wnt signaling and PLD is not known.Here, we demonstrate that PLD isozymes, PLD1 and PLD2 are direct targets and positive feedback regulators of the Wnt/β-catenin signaling. Wnt3a and Wnt mimetics significantly enhanced the expression of PLDs at a transcriptional level in HCT116 colorectal cancer cells, whereas silencing of β-catenin gene expression or utilization of a dominant negative form of T cell factor-4 (TCF-4) inhibited expression of PLDs. Moreover, both PLD1 and PLD2 were highly induced in colon, liver and stomach tissues of mice after injection of LiCl, a Wnt mimetic. Wnt3a stimulated formation of the β-catenin/TCF complexes to two functional TCF-4-binding elements within the PLD2 promoter as assessed by chromatin immunoprecipitation assay. Suppressing PLD using gene silencing or selective inhibitor blocked the ability of β-catenin to transcriptionally activate PLD and other Wnt target genes by preventing formation of the β-catenin/TCF-4 complex, whereas tactics to elevate intracellular levels of phosphatidic acid, the product of PLD activity, enhanced these effects. Here we show that PLD is necessary for Wnt3a-driven invasion and anchorage-independent growth of colon cancer cells.PLD isozyme acts as a novel transcriptional target and positive feedback regulator of Wnt signaling, and then promotes Wnt-driven anchorage-independent growth of colorectal cancer cells. We propose that therapeutic interventions targeting PLD may confer a clinical benefit in Wnt/β-catenin-driven malignancies. Colorectal cancer is one of the most common malignancies, occurring in a significant percentage of the population. More than 80% of sporadic and hereditary colorectal cancers may be caused by aberrations in the Wnt/β-catenin signaling pathway Phospholipase D (PLD) catalyzes hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA), which acts as a second messenger in many physiological responses Among one of the genes identified in this screen was PLD1, and suppression of PLD1 significantly inhibited both β-catenin transcriptional activity and colon cancer cell proliferation. In the present study, we demonstrate the action of PLD isozymes as novel targets and positive feedback regulators of Wnt signaling. Thus, identification of a Wnt-β-catenin-TCF-regulated PLD axis provides new mechanistic insights into cancer.Human colorectal cancer cells  and breast cancer cells (HS578T) were purchased from ATCC  and were grown according to standard protocols. Purified recombinant Wnt3a was purchased from R&D Systems Inc. BIO was obtained from Calbiochem. LiCl, 1- or 3-butanol, dioctanoyl PA, and 1-propranolol were purchased from Sigma-Aldrich. PLD1 and PLD2 selective inhibitors were purchased from Cayman chemical. Dual luciferase assay kits were purchased from Promega.Human PLD1 (pGL4-PLD1 Luc) and PLD2 (pGL4-PLD2 Luc) promoter reporter plasmids contain −1.9 kb (−1930/+1) and 2.6 kb (−2180/+491) of upstream 5′-flanking DNA linked to luciferase reporter genes, respectively, and have been described elsewhere AAGGUGGGACGACAAUGAGCA) and human PLD2 .Mutations of TCF-4 binding elements on the PLD2 promoter were generated using the Quick Change Site-Directed Mutagenesis Kit, according to the manufacturer's recommendations  or Polyfect (Qiagen) reagents. Transfection and luciferase assays were performed as previously described Cell lysates were analyzed for immunoprecipitation and/or immunoblot, as previously described 3H] phosphatidylbutanol, the product of PLD-mediated transphosphatidylation, in the presence of 1-butanol, as previously described PLD activity was assessed by measurement of the formation of [Total RNA (1 µg) was pretreated with DNase and used for reverse transcription with M-MLV reverse transcriptase (Invitrogen). Real-time Q-PCR was performed on a Rotor-Gene RG-6000A apparatus (Corbett Research): for 44 cycles of 94°C for 10 sec, 60°C for 10 sec, and 72°C for 15 sec. Reactions (20 µl) included 2 µl of cDNA, target-specific primers, and the Quantitect SYBR green PCR kit (QIAGEN). The temperature range for analysis of melting curves was 55°C to 99°C over 30 sec. Three independent experiments were performed for each reaction in triplicate. All data were normalized with GAPDH gene expression values. See 2 and stored at −70°C for immunoblotting. Mice (n = 3/group) also were anesthetized and then perfused intracardially with 4% paraformaldehyde in PBS containing 0.34% L-lysine (Sigma). Following fixative perfusion, the tissues were removed, placed in 4% paraformaldehyde solution at 4°C overnight, and then transferred to a 30% sucrose solution. The tissues were processed by coronal cryostat sectioning in Dulbecco's PBS solution containing 0.1% sodium azide using a freezing microtome .Mice were provided with standard maintenance and a diet from Dae Han Bio Link . Animal studies were approved and performed under guidelines of the Institute of Health Guidelines for the Institutional Review Board of Pusan National University . Twelve week-old male FVB mice received intravenous injection with LiCl once each day for 2 days at dose of 5 mg/kg. The control group was injected with the same volume of phosphate-buffered saline (PBS). Forty-eight hrs after LiCl injection, animals (n = 3/group) were deeply anesthetized with diethyl ether and decapitated, and the tissues were frozen immediately in LN® . Two-color fluorescent image for anti-PLD and β-catenin antibody staining were collected on a Zeiss LSM 510 confocal microscope . Fluorescent images were analyzed using Zeiss LSM image browser software (Zeiss).Paraformaldehyde-fixed 4 µm paraffin sections of colon tissues were autoclaved in 10 mM sodium citrate buffer (pH 6.0). After blocking with 5% BSA and 1% normal goat serum in PBS, sections were incubated with anti-β-catenin (BD transduction) and PLD antibodies overnight at 4°C, followed by washing with PBS. Subsequently, sections were incubated with Alexa fluor 488 or Alexa fluor 555-conjugated IgG secondary antibody  at room temperature for 1 h, followed by washing with PBS. After counterstaing with DAPI, and slides were mounted in PermountChIP experiments were performed as previously described Cells were added to Transwell membrane chambers . The number of cells that migrated through the membrane to the lower chamber was counted after 24 hours. For siRNA experiments, cells were seeded 24 hours after transfection with siRNAs, and migration assays were performed for another 24 hours. For the invasion assays, Matrigel  was added to Transwell membrane chambers and incubated for 5 hours; cells were then seeded. Extent of migration and invasion were expressed as an average number of cells per microscopic field.In Vitro Tumor Sensitivity Assay , according to the manufacturer's specifications. Anchorage independent growth was examined in soft agar; 50 µl of base agar matrix was added to the bottom of each well of a 96-well plate. Once the agar was solid, 75 µl of cell suspension/soft agar matrix containing 3×103 cells was layered onto the top, followed by 50 µl of 2× complete medium with Wnt3a and/or inhibitors. After 10 days of incubation, the agar matrix was solubilized, and 3--2,5-diphenyl tetrazolium bromide was added to each well. The absorbance produced by formation of insoluble formazan product by viable cells was recorded at 570 nm.Anchorage independent growth was measured using the CytoSelect™ 96-Well t test, and P<0.05 was considered statistically significant.All experiments were independently performed at least three times, with similar results. Data were analyzed by the Student's To examine the question of whether or not Wnt signaling increases PLD expression, HCT116 colorectal cancer cells were exposed to Wnt3a or Wnt3a mimetics . As detein vivo. LiCl was injected into mice, and expression of PLD was investigated in several tissues using anti-PLD antibody which recognizes both PLD1 and PLD2. We have reported on the specificity of the antibody to PLD To further strengthen our results, we investigated the effect of blockade of GSK3β on PLD expression We examined the question of whether or not expression of PLD isozymes is indeed transcriptionally activated by β-catenin or TCF-4. As shown in A polymorphism of the PLD2 gene has recently been associated with prevalence of colorectal carcinoma As shown in As shown in To examine the functional importance of the TBE motif in regulation of PLD2 gene transcription, site-directed mutation of TBE sites was generated in the context of a PLD2 promoter. Mutation in the TBE1 or TBE2 site significantly decreased Wnt3a-induced PLD2 promoter activity in HCT116 cells . Mutatioin vivo binding of β-catenin/TCF-4 to the PLD2 promoter. DNA-β-catenin/TCF-4 complexes were immunoprecipitated with antibodies shown in Furthermore, we then performed a chromatin immunoprecipitation (ChIP) assay in HCT116 cells to confirm We investigated the question of whether or not Wnt signaling-induced PLDs upregulation might modulate β-catenin-dependent TCF transcriptional activity via a positive feedback loop. Ectopic expression of a constitutive active mutant of β-catenin increased TCF transcriptional activity in HCT116 colon cancer cells . Selectiin vitro tumor sensitivity assay, we further investigated the question of whether or not PLD isozyme is required for Wnt/β-catenin-mediated tumorigenic effects. Treatment with Wnt3a in HCT116 cells increased anchorage-independent colony growth, whereas depletion and selective inhibitor of the PLD isoform significantly suppressed Wnt3a-driven anchorage-independent growth  and can enhance proliferation, migration, and invasion The present finding describing PLD isozyme as a new target and positive regulator of Wnt/β-catenin further expands understanding of the existing and rather extensive regulatory network of the Wnt signaling pathway. Accordingly, therapeutic interventions that target the PLD enzymatic activity using PLD inhibitor may be of clinical value in colorectal cancer. Future studies are warranted for further examination of the role of the Wnt/β-catenin/PLD pathway in cancer development.Figure S1Wnt3a increases in a time dependent manner mRNA levels of PLD isozymes in HCT116 cells. The purified recombinant Wnt3a (150 ng/ml) was treated in HCT116 cells for the indicated times, and the expression level of PLD isozymes were analyzed by Q-RT-PCR. *P<0.05 compared with non-treatment. Data represent the mean ± S.D. of three independent experiments.(0.03 MB PDF)Click here for additional data file.Figure S2Wnt-dependent increase of PLD mRNA is due to elevated transcription. HCT116 cells were pretreated with actinomycin D (5 µg/ml) and treated with Wnt3a (150 ng/ml) for the indicated times and then PLD mRNA levels were analyzed by Q-RT-PCR. *P<0.05 compared with non-treatment; **P<0.05 compared with Wnt3a. Data represent the mean ± S.D. of three independent experiments.(0.04 MB PDF)Click here for additional data file.Figure S3Wnt3a increases mRNA levels of PLD isozymes in a variety of cancer cells. HCA-7, Colo-741, RKO and HS578T cells were stimulated by Wnt3a (150 ng/ml) for 12 h. Expression of PLD isozymes were analyzed by Q-RT-PCR. *P<0.05 versus vehicle. Data represent the mean ± S.D. of three independent experiments.(0.04 MB PDF)Click here for additional data file.Figure S4Effect of PLD siRNAs on expression of PLD isozymes. HCT116 cells were transfected with siRNAs for control or PLD isozyme and the expression of PLD isozymes was analyzed by Q-RT-PCR and immunoprecipitation/immunoblotting using antibody to PLD. *P<0.05 versus control-siRNA.(0.08 MB PDF)Click here for additional data file.Figure S5PLD activity is required for Wnt-induced β-catenin/TCF-4 association. HCT116 cells were pretreated with 1- or 3-butanol (0.6%) and stimulated with Wnt3a (150 ng/ml) for 24 h. Association of TCF-4 with β-catenin was analyzed by immunoprecipitation and immunoblot using the indicated antibodies. Protein levels were determined by immunoprecipitation or immunoblotting using the indicated antibodies. Interaction levels or protein expression were quantitated by densitometer analysis. Data are representative of three independent experiments.(0.08 MB PDF)Click here for additional data file.Table S1Primer sets for deletion constructs of the hPLD2 promoter region.(0.03 MB DOC)Click here for additional data file.Table S2Consensus TBE in the PLD2 promoter.(0.04 MB DOC)Click here for additional data file.Table S3Primer sets for Q-RT-PCR.(0.04 MB DOC)Click here for additional data file.Table S4Primer sets for ChIP assay.(0.03 MB DOC)Click here for additional data file.


Interestingly, ST6Gal-I mRNA is known to be upregulated in metastatic ovarian cancer, therefore the goal of the present study was to determine whether ST6Gal-I confers a similarly aggressive phenotype to ovarian tumor cells.Ovarian adenocarcinoma is not generally discovered in patients until there has been widespread intraperitoneal dissemination, which is why ovarian cancer is the deadliest gynecologic malignancy. Though incompletely understood, the mechanism of peritoneal metastasis relies on primary tumor cells being able to detach themselves from the tumor, escape normal apoptotic pathways while free floating, and adhere to, and eventually invade through, the peritoneal surface. Our laboratory has previously shown that the Golgi glycosyltransferase, ST6Gal-I, mediates the hypersialylation of βThree ovarian carcinoma cell lines were screened for ST6Gal-I expression, and two of these, PA-1 and SKOV3, were found to produce ST6Gal-I protein. The third cell line, OV4, lacked endogenous ST6Gal-I. In order to understand the effects of ST6Gal-I on cell behavior, OV4 cells were stably-transduced with ST6Gal-I using a lentiviral vector, and integrin-mediated responses were compared in parental and ST6Gal-I-expressing cells.Forced expression of ST6Gal-I in OV4 cells, resulting in sialylation of β1 integrins, induced greater cell adhesion to, and migration toward, collagen I. Similarly, ST6Gal-I expressing cells were more invasive through Matrigel.ST6Gal-I mediated sialylation of β1 integrins in ovarian cancer cells may contribute to peritoneal metastasis by altering tumor cell adhesion and migration through extracellular matrix. N-acetyllactosamine structures  is a Golgi-mediated process facilitated by the enzyme, β-galactoside α2–6-sialyltransferase . Variant α2–6 sialylation can have a wide array of biologic and pathogenic consequences, including alterations in immune response and embryogenesis, as well as a role in the development and progression of some cancers [The α2–6 linkage of sialic acids to ures Galβ–4GlcNAc  cancers . Perturb1 integrin. Though incompletely understood, β1 hypersialylation could modify integrin-dependent cell responses through a change in receptor conformation, by masking functional domains within the integrin heterodimer, by affecting integrin interaction with other membrane bound proteins or glycolipids, or by another, as yet, unrecognized mechanism [The overexpression of ST6Gal-I is well documented in several diverse cancer types. These cancers include: colorectal , cervicaechanism . Lin andechanism . Conversechanism . Taken iechanism ,6, thougechanism ,17.The role of ST6Gal-I in ovarian carcinoma has not been as clearly defined as its effect in some other tumors, namely colon and breast. Nonetheless, there are recent data indicative of the emerging attention to the importance of sialylation in ovarian cancer. High-throughput techniques have yielded evidence that ST6Gal-I is up-regulated in epithelial ovarian malignancy. For example, proteomic analysis revealed α2–6 sialylation to be proportionally favored over α2–3 sialylation . This mi1 integrin hypersialylation, induced increased adhesion to collagen I, migration toward collagen I, and invasiveness through Matrigel. Our results suggest a potential role for variant sialylation in the dissemination of ovarian carcinoma.In the present study, we screened three separate ovarian carcinoma cell lines for endogenous expression of ST6Gal-I, and found that two of these were positive for ST6Gal-I protein. The third, the OV4 cell line, had negligible levels of the enzyme and therefore, to assess the effects of α2–6 sialylation on promoting the tumor cell phenotype, we forced ST6Gal-I expression and evaluated integrin-dependent cell behaviors. ST6Gal-I expression, with consequent β2 and passaged two to three times per week.The ovarian carcinoma cell line SKOV3 was generously gifted to us by Dr. Janet Price , whereas the OV4 cell line was a generous gift from Dr. Timothy Eberlein . The PA1 cell line was purchased commercially through ATCC . PA1 cells were cultured and grown in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum  and penicillin, streptomycin, and amphotericin B. OV4 and SKOV3 cells were cultured and grown in Dulbecco's modified Eagle's MEM/Ham's F-12 50:50 (DMEM/F12) supplemented with 10% FBS, penicillin, streptomycin, and amphotericin B. Cells were maintained at 37°C in 5% CO1 integrin , or the V5 epitope . Membranes were then washed and incubated with horseradish peroxidase-coupled secondary antibody . The labeled proteins were visualized with enhanced chemiluminescence, and subsequent images were scanned with a Hewlett-Packard Scanjet 5470 c .Cells were lysed in buffer composed of 50 mM Tris-HCl (pH 7.4) containing 1% Triton X-100, and a protease inhibitor cocktail (Roche Applied Bioscience). Protein concentrations of the lysates were determined using a modified Bradford Assay . Proteins were resolved by reducing SDS-PAGE, and transferred to polyvinylidene difluoride membranes. Membranes were blocked with 5% nonfat dry milk in TBS containing 0.05% Tween 20 (TBST). Primary antibodies were then added to the membranes for incubation, with antibody against ST6Gal-I , β1 integrin.Cell lysates were incubated overnight at 4°C with rotation with 100 μg/mL of the α2–6 sialic acid-specific lectin, SNA-1, conjugated to agarose beads . The lectin-glycoprotein complexes were collected by centrifugation, washed with lysis buffer, and released from the bead complexes by boiling in SDS-PAGE sample buffer. Precipitated proteins were resolved by reducing SDS-PAGE, and immunoblotted to detect βAn ST6Gal-I cDNA construct, containing a C-terminal V5 tag, was a generous gift from Dr. Karen Colley . This construct was incorporated into a lentiviral vector containing a puromycin-resistance cassette for selection of stably-transduced cells, as previously described . OV4 cel4 cells were plated onto culture dishes pretreated with 20 μg/mL bovine collagen I and blocked with 2% denatured bovine serum albumin (dBSA). To control for nonspecific binding, cells were also plated onto dishes pretreated with dBSA alone. Cells were allowed to adhere for 30 minutes at 37°C, and then samples were washed gently with PBS. The remaining adherent cells were fixed using formaldehyde and 4% sucrose, and subsequently stained with crystal violet and solubilized with 10% acetic acid. Absorbance of the solution dye was measured at 540 nm.The parental (P), ST6Gal-I-expressing (ST6), and empty vector-transduced (EV) cells were cultured in serum-free DMEM/F12 media for 48 hours. Cells were disengaged from the culture flasks using CellStripper solution  and 8 × 105cells were then seeded into the upper wells of Boyden chambers included in the QCM Collagen I Quantitative Cell Migration Assay Kit . The chambers were lined with 8.0 μm polyethylene terpthalate (PET) membranes coated on the underside with a collagen I concentration gradient. To control for nonspecific migration, cells were also seeded into Boyden chambers with PET membranes coated with BSA. The lower chambers contained 300 μL of conditioned, serum-free NIH3T3 media for the chemoattractant. Cells were allowed to incubate at 37°C for 14 hours, and migration to the underside of the membrane was quantified as per the vendor's staining protocol.P, ST6, and EV cells were cultured in serum-free media for 48 hours and disengaged from the culture dishes using CellStripper solution. 2.5 × 105 cells were seeded into the upper wells of Boyden chambers lined with 8.0 μm PET membranes with a thin layer of GFR Matrigel Basement Membrane Matrix. The lower chamber contained 300 μL of conditioned, serum-free NIH3T3 media for the chemoattractant. Cells were incubated at 37°C for 48 hours, and invasion was quantified as per the vendor's staining protocol.P, ST6, and EV cells were cultured in serum-free DMEM/F12 media for 48 hours prior to being disengaged from the culture flasks using CellStripper solution. BD BioCoat Growth Factor Reduced (GFR) Matrigel Invasion Chamber  assay kits were used to measure invasion. 5 × 10in vitro or in vivo. We chose three established ovarian carcinoma cell lines to screen for the enzyme: PA1, OV4, and SKOV3. To this end, cells were lysed and immunoblotted for ST6Gal-I. As shown in Fig. Levels of ST6Gal-I mRNA have been shown to be increased in ovarian carcinoma , but, to1integrin, we evaluated integrin reactivity to SNA-1, a lectin which specifically recognizes α2–6-linked sialic acids. Briefly, cell lysates were incubated with agarose-conjugated SNA-1, and SNA-bound glycoproteins were then collected by centrifugation. The glycoproteins were resolved by SDS-PAGE, and Western blotted for β1 integrin  displayed variable electrophoretic mobility for the three cell lines, with the bands from PA1 and SKOV3 cells showing reduced mobility. As we have previously reported, changes in electrophoretic mobility of the mature β1 integrin isoform typically reflect variation in the degree of α2–6 sialylation [1 immunoblots is thought to represent a precursor integrin isoform localized to the endoplasmic reticulum, and as such, is not a substrate for ST6Gal-I. The precursor isoform was not observed in OV4 cells.To assess levels of α2–6 sialylation on the ST6Gal-I substrate, βrin Fig. . In linenes Fig. . Intereslylation ,21. ThusIn order to illustrate the role of α2–6 sialylation in modifying integrin-dependent cell behaviors, OV4 cells were stably transduced with a lentiviral vector containing a V5-tagged ST6Gal-I construct (ST6). An empty-vector control cell line (EV) was also generated (note that these cell lines represent a pooled population of stably-transduced clones). Expression of the ST6Gal-I construct was confirmed by Western blotting for both ST6Gal-I and for the V5 tag Fig. . Neither1 integrin, and, as expected, only the β1 integrins from ST6Gal-I expressing cells were found to be α2–6 sialylated , which confers a worse prognosis than if the disease were discovered at an earlier stage. Though the process of peritoneal seeding is poorly understood, the most widely accepted hypothesis is that cells detach from the primary tumor, and are transported via peritoneal fluid throughout the abdomen, eventually attaching themselves to the peritoneal surface. Phenotypically, the tumor cells with the best chance of metastasizing are cells with the ability to escape apoptosis following detachment, while exhibiting increased capacity to adhere to, and invade through the peritoneum, which is exactly the cellular phenotype routinely seen in advanced stage ovarian carcinoma . In the  cancers ,12.in vivo [in vitro [in vitro [2) decreases ST6Gal-I expression in a dose dependent fashion in the human breast cancer cell line, MCF-7, an effect reversed with Tamoxifen [2 in ovarian cancers has been demonstrated in SKOV3 to be due to a mutation in estrogen receptor-α [1 integrin receptor.The regulation of ST6Gal-I expression is multifactorial. Its expression is increased by oncogenic ras -11, thouin vivo ,30, and in vitro . Furtherin vitro . Estradiamoxifen . A lack ceptor-α , and thu1 integrins expressed by colon tumor cells increases cell adhesion to, and migration on collagen I [in vitro by LP9 mesothelial cells, along with fibronectin, laminin, vitronectin, and collagen types III and IV. In vivo, these molecules would contribute to the make up of the extracellular matrix (ECM) that free floating ovarian carcinoma cells would encounter, adhere to, and subsequently invade [1 integrin's importance in the metastasis of ovarian cancer has been repeatedly demonstrated. β1 integrin is integral to multicellular spheroid formation [1 function have focused on either changes in integrin expression or regulation of activity through "inside-out" signaling mechanisms . However, there is growing appreciation for the role of variant sialylation in modulating β1 activity.We have previously shown that ST6Gal-I-mediated sialylation of βllagen I . Likewisy invade . β1 inteormation , adhesioormation ,37, migrormation , and sphormation . Most stGiven the extensive evidence of hypersialylation in tumor progression, sialyltransferases have been investigated as potential targets for drug therapy . ST6Gal-1 integrins. Overexpression of ST6Gal-I has previously been implicated in colorectal and breast adenocarcinomas, however, only limited information has been available regarding the role of this enzyme in ovarian cancer. The accumulating evidence indicating that ST6Gal-I-mediated integrin sialylation causes increased cell migration and invasion in multiple tumor types suggests that ST6Gal-I is a promising target for therapeutic intervention.In this study, we have shown that cell behaviors consistent with a metastatic phenotype can be induced in ovarian tumor cells by upregulation of ST6Gal-I, with consequent α2–6 sialylation of βDRC, FMS and JAL IV were directly involved in data acquisition and analysis. DRC wrote the manuscript with editorial assistance from JAL III and SLB. DRC, SLB and JAL III were responsible for the initial conception and design of the study.