Pax9's dual roles in modulating Wnt signaling during murine palatogenesis.

BACKGROUND: Despite the strides made in understanding the complex network of key regulatory genes and cellular processes that drive palate morphogenesis, patients suffering from these conditions face treatment options that are limited to complex surgeries and multidisciplinary care throughout life. Hence, a better understanding of how molecular interactions drive palatal growth and fusion is critical for the development of treatment and preventive strategies for cleft palates in humans. Our previous work demonstrated that Pax9-dependent Wnt signaling is critical for the growth and fusion of palatal shelves. We showed that controlled intravenous delivery of small molecule Wnt agonists specifically blocks the action of Dkks (inhibitors of Wnt signaling) and corrects secondary palatal clefts in Pax9-/- mice. While these data underscore the importance of the functional upstream relationship of Pax9 to the Wnt pathway, not much is known about how the genetic nature of Pax9's interactions in vivo and how it modulates the actions of these downstream effectors during palate formation. RESULTS: Here, we show that the genetic reduction of Dkk1 during palatogenesis corrected secondary palatal clefts in Pax9-/- mice with restoration of Wnt signaling activities. In contrast, genetically induced overexpression of Dkk1 mice phenocopied the defects in tooth and palate development visible in Pax9-/- strains. Results of ChIP-qPCR assays showed that Pax9 can bind to regions near the transcription start sites of Dkk1 and Dkk2 as well as the intergenic region of Wnt9b and Wnt3 ligands that are downregulated in Pax9-/- palates. CONCLUSIONS: Taken together, these data suggest that the molecular mechanisms underlying Pax9's role in modulating Wnt signaling activity likely involve the inhibition of Dkk expression and the control of Wnt ligands during palatogenesis.

ß-catenin is required for taste bud cell renewal and behavioral taste perception in adult mice.

Taste stimuli are transduced by taste buds and transmitted to the brain via afferent gustatory fibers. Renewal of taste receptor cells from actively dividing progenitors is finely tuned to maintain taste sensitivity throughout life. We show that conditional ß-catenin deletion in mouse taste progenitors leads to rapid depletion of progenitors and Shh+ precursors, which in turn causes taste bud loss, followed by loss of gustatory nerve fibers. In addition, our data suggest LEF1, TCF7 and Wnt3 are involved in a Wnt pathway regulatory feedback loop that controls taste cell renewal in the circumvallate papilla epithelium. Unexpectedly, taste bud decline is greater in the anterior tongue and palate than in the posterior tongue. Mutant mice with this regional pattern of taste bud loss were unable to discern sweet at any concentration, but could distinguish bitter stimuli, albeit with reduced sensitivity. Our findings are consistent with published reports wherein anterior taste buds have higher sweet sensitivity while posterior taste buds are better tuned to bitter, and suggest ß-catenin plays a greater role in renewal of anterior versus posterior taste buds.

Wnt signaling regulates homeostasis of the periodontal ligament.

BACKGROUND AND OBJECTIVE: In health, the periodontal ligament maintains a constant width throughout an organism's lifetime. The molecular signals responsible for maintaining homeostatic control over the periodontal ligament are unknown. The purpose of this study was to investigate the role of Wnt signaling in this process by removing an essential chaperone protein, Wntless (Wls), from odontoblasts and cementoblasts, and observing the effects of Wnt depletion on cells of the periodontal complex. MATERIAL AND METHODS: The Wnt responsive status of the periodontal complex was assessed using two strains of Wnt reporter mice: Axin2(LacZ/+) and Lgr5(LacZ/+) . The function of this endogenous Wnt signal was evaluated by conditionally eliminating the Wntless (Wls) gene using an osteocalcin Cre driver. The resulting OCN-Cre;Wls (fl/fl) mice were examined using micro-computed tomography and histology, immunohistochemical analyses for osteopontin, Runx2 and fibromodulin, in-situ hybridization for osterix and alkaline phosphatase activity. RESULTS: The adult periodontal ligament is Wnt responsive. Elimination of Wnt signaling in the periodontal complex of OCN-Cre;Wls(fl/fl) mice resulted in a wider periodontal ligament space. This pathologically increased periodontal width is caused by a reduction in the expression of osteogenic genes and proteins, which results in thinner alveolar bone. A concomitant increase in fibrous tissue occupying the periodontal space was observed, along with a disruption in the orientation of the periodontal ligament. CONCLUSION: The periodontal ligament is a Wnt-dependent tissue. Cells in the periodontal complex are Wnt responsive, and eliminating an essential component of the Wnt signaling network leads to a pathological widening of the periodontal ligament space. Osteogenic stimuli are reduced, and a disorganized fibrillary matrix results from the depletion of Wnt signaling. Collectively, these data underscore the importance of Wnt signaling in homeostasis of the periodontal ligament.

Impact of genetic variations in the WNT family members and RUNX2 on dental and skeletal maturation: a cross-sectional study.

BACKGROUND: This study evaluated if genetic variations in the WNT family members and RUNX2 are associated with craniofacial maturation, investigating dental and skeletal maturity in children and teenagers. METHODS: Radiographs from pre-orthodontic treatment of Brazilian patients (7 to 17 years-old) were used to assess dental (panoramic radiographs) and skeletal maturity (cephalometric radiographs). The chronological age (CA) was calculated based on the date of birth and the time the radiographs were performed. For the dental maturity analysis, the Demirjian (1973) method was used and a delta [dental age - chronological age (DA-CA)] was calculated. For the skeletal maturity analysis, the Baccetti et al. (2005) method was used and the patients were classified as "delayed skeletal maturation", "advanced skeletal maturation" or "normal skeletal maturation". DNA isolated from buccal cells was used for genotyping of two genetic variations in WNT family genes: rs708111 (G > A) in WNT3A and rs1533767 (G > A) in WNT11; and two genetic variations in RUNX2: rs1200425 (G > A) and rs59983488 (G > T). A statistical analysis was performed and values of p < 0.05 indicated a significant difference. RESULTS: There were no associations between dental maturity and genotypes (p > 0.05). In the skeletal maturity analysis, the allele A in the rs708111 (WNT3A) was statistically more frequent in patients with delayed skeletal maturation (Prevalence Ratio = 1.6; 95% Confidence Interval = 1.00 to 2.54; p-value = 0.042). CONCLUSIONS: The rs708111 in the WNT3A gene impacts on skeletal maturation.

Differential expression of canonical and non-canonical Wnt ligands in ameloblastoma.

BACKGROUND: Canonical and non-canonical Wnt signaling pathways modulate diverse cellular processes during embryogenesis and post-natally. Their deregulations have been implicated in cancer development and progression. Wnt signaling is essential for odontogenesis. The ameloblastoma is an odontogenic epithelial neoplasm of enamel organ origin. Altered expressions of Wnts-1, -2, -5a, and -10a are detected in this tumor. The activity of other Wnt members remains unclarified. MATERIALS AND METHODS: Canonical (Wnts-1, -2, -3, -8a, -8b, -10a, and -10b), non-canonical (Wnts-4, -5a, -5b, -6, 7a, -7b, and -11), and indeterminate groups (Wnts-2b and -9b) were examined immunohistochemically in 72 cases of ameloblastoma (19 unicystic [UA], 35 solid/multicystic [SMA], eight desmoplastic [DA], and 10 recurrent [RA]). RESULTS: Canonical Wnt proteins (except Wnt-10b) were heterogeneously expressed in ameloblastoma. Their distribution patterns were distinctive with some overlap. Protein localization was mainly membranous and/or cytoplasmic. Overexpression of Wnt-1 in most subsets (UA = 19/19; SMA = 35/35; DA = 5/8; RA = 7/10) (P < 0.05), Wnt-3 in granular cell variant (n = 3/3), and Wnt-8b in DA (n = 8/8) was key observations. Wnts-8a and -10a demonstrated enhanced expression in tumoral buddings and acanthomatous areas. Non-canonical and indeterminate Wnts were absent except for limited Wnt-7b immunoreactivity in UA (n = 1/19) and SMA (n = 1/35). Stromal components expressed variable Wnt positivity. CONCLUSION: Differential expression of Wnt ligands in different ameloblastoma subtypes suggests that the canonical and non-canonical Wnt pathways are selectively activated or repressed depending on the tumor cell differentiation status. Canonical Wnt pathway is most likely the main transduction pathway while Wnt-1 might be the key signaling molecule involved in ameloblastoma tumorigenesis.

Studies with Wnt genes and nonsyndromic cleft lip and palate.

BACKGROUND: Clefts of the lip and/or palate (cleft lip/palate) are notable for their complex etiology. The WNT pathway regulates multiple developmental processes including craniofacial development and may play a role in cleft lip/palate and other defects of craniofacial development such as tooth agenesis. Variations in WNT genes have been recently associated with cleft lip/palate in humans. In addition, two WNT genes, Wnt3 and Wnt9B, are located in the clf1 cleft locus in mice. METHODS: We investigated 13 SNPs located in Wnt3A, Wnt5A, Wnt8A, Wnt11, Wnt3, and Wnt9B genes for association with cleft lip/palate subphenotypes in 463 cleft cases and 303 unrelated controls. Genotyping of selected polymorphisms was carried out using Taqman assays. PLINK 1.06 software was used to test for differences in allele frequencies of each polymorphism between affected and unaffected individuals. Haplotype analysis was also performed. RESULTS: Individuals carrying variant alleles in WNT3 presented an increased risk for cleft lip/palate (p = 0.0003; OR, 1.61; 95% CI, 1.29-2.02) in the population studied. CONCLUSION: Our results continue to support a role for WNT genes in the pathogenesis of cleft lip/palate. Although much remains to be learned about the function of individual WNT genes during craniofacial development, additional studies should focus on the identification of potentially functional variants in these genes as contributors to human clefting. Birth Defects Research (Part A), 2010. © 2010 Wiley-Liss, Inc.

Cross-talk between the TGFbeta and Wnt signaling pathways in murine embryonic maxillary mesenchymal cells.

The transforming growth factor beta (TGFbeta) and Wnt signaling pathways play central roles regulating embryogenesis and maintaining adult tissue homeostasis. TGFbeta mediates its cellular effects through types I and II cell surface receptors coupled to the nucleocytoplasmic Smad proteins. Wnt signals via binding to a cell surface receptor, Frizzled, which in turn activates intracellular Dishevelled, ultimately leading to stabilization and nuclear translocation of beta-catenin. Previous studies have demonstrated several points of cross-talk between the TGFbeta and Wnt signaling pathways. In yeast two-hybrid and GST-pull down assays, Dishevelled-1 and Smad 3 have been shown to physically interact through the C-terminal one-half of Dishevelled-1 and the MH2 domain of Smad 3. The current study demonstrates that co-treatment of murine embryonic maxillary mesenchyme (MEMM) cells with Wnt-3a and TGFbeta leads to enhanced reporter activity from TOPflash, a Wnt-responsive reporter plasmid. Transcriptional cooperation between TGFbeta and Wnt did not require the presence of a Smad binding element, nor did it occur when a TGFbeta-responsive reporter plasmid (p3TP-lux) was transfected. Overexpression of Smad 3 further enhanced the cooperation between Wnt and TGFbeta while overexpression of dominant-negative Smads 2 and 3 inhibited this effect. Co-stimulation with TGFbeta led to greater nuclear translocation of beta-catenin, providing explanation for the effect of TGFbeta on Wnt-3a reporter activity. Wnt-3a exerted antiproliferative activity in MEMM cells, similar to that exerted by TGFbeta. In addition, Wnt-3a and TGFbeta in combination led to synergistic decreases in MEMM cell proliferation. These data demonstrate a functional interaction between the TGFbeta and Wnt signaling pathways and suggest that Wnt activation of the canonical pathway is an important mediator of MEMM cell growth.

Expression patterns of WNT/ß-CATENIN signaling molecules during human tooth development.

The WNT/ß-CATENIN signaling has been demonstrated to play critical roles in mouse tooth development, but little is known about the status of these molecules in human embryonic tooth. In this study, expression patterns of WNT/ß-CATENIN signaling components, including WNT ligands (WNT3, WNT5A), receptors (FZD4, FZD6, LRP5), transducers (ß-CATENIN), transcription factors (TCF4, LEF1) and antagonists (DKK1, SOSTDC1) were investigated in human tooth germ at the bud, cap and bell stages by in situ hybridization. All these genes exhibited similar but slightly distinct expression patterns in human tooth germ in comparison with mouse. Furthermore the mRNA expression of these genes in incisors and molars at the bell stage was also examined by real-time PCR. Our results reveal the status of active WNT/ß-CATENIN signaling in the human tooth germ and suggest these components may also play an essential role in the regulation of human tooth development.

[Expression of wingless-type MMTV integration site family member 3 in rat dental follicle tissues and cells undergoing osteogenic differentiation].

OBJECTIVE: To investigate the expression of wingless-type MMTV integration site family, member 3 (Wnt3) in rat dental follicles and its protein level in dental follicle cells (DFC) undergoing osteogenic induction and to discuss the effects of Wnt3 on the differentiation of DFC. METHODS: Rats at postnatal days 1, 3, 5, 7, 9, 11 and 13 were executed, then the mandibles were immediately removed and immunohistochemistry was performed to detect the expression of Wnt3 in dental follicles of postnatal rats. The expression and distribution of Wnt3 in DFC were determined by immunofluorescence. Alizarin red-S staining was performed to assess the mineralization of DFC. Western blotting was used to evaluate Wnt3 and ß-catenin protein levels after stimulated by osteogenic medium for 1, 2 and 3 weeks, respectively. RESULTS: Immunohistochemistry revealed that the expression of Wnt3 in rat dental follicles began at day 5 and sustained to day 13. On day 1 and 3, the expression of Wnt3 in dental follicles was negative.Wnt3 was expressed in the cytoplasm of DFC. Alizarin red-S staining indicated that the osteogenic medium stimulated the differentiation of DFC into osteoblastic lineage.Western blotting demonstrated that the Wnt3 protein levels were significantly up-regulated after stimulated with osteogenic medium for 1 weeks compared with the control (2.60 ± 0.04 vs.1.00 ± 0.00, P < 0.05). Then the levels of Wnt3 protein were declined, and at the 3rd week, no significant difference was observed between osteo-induced group and the control (1.00 ± 0.05 vs.1.00 ± 0.00, P > 0.05). The levels of ß-catenin were increased in osteo-induced groups compared with the control (1.95 ± 0.05 vs.1.00 ± 0.00, P < 0.05; 9.77 ± 0.65 vs.1.00 ± 0.00, P < 0.05;1.75 ± 0.21 vs.1.00 ± 0.00, P < 0.05). Furthermore, the expression of ß-catenin reached to a peak on the 2nd week (9.77 ± 0.65), and then declined. CONCLUSIONS: Wnt3 was expressed in the rat dental follicles both in vivo and in vitro and up-regulated during early phase of osteoblast differentiation in DFC.Wnt3 may be involved in early phase of osteoblast differentiation.

Mechanical loading activates ß-catenin signaling in periodontal ligament cells.

OBJECTIVE: To determine whether ß-catenin signaling is responsive to mechanical loading in periodontal ligament (PDL) cells. MATERIALS AND METHODS: To determine whether Wnt/ß-catenin signaling pathway components are present and functional, PDL cells were treated with lithium chloride or Wnt3a-conditioned media. To determine whether mechanical strain activates ß-catenin signaling, PDL cells were subjected to compressive loading. Activation of the ß-catenin signaling pathway was determined by immunofluorescence, Western immunoblotting, and TOPflash assay. RESULTS: Mimicking Wnt signaling stimulates ß-catenin nuclear translocation and T-cell factor/lymphoid enhancer binding factor-dependent transcriptional activation in PDL cells. Mechanical loading stimulates a transient accumulation of dephosphorylated ß-catenin in the cytoplasm and its translocation to the nucleus. This effect of strain acts through activation of protein kinase B and phosphorylation of glycogen synthase kinase-3 beta. These strain-related changes do not involve the low-density lipoprotein receptor-related protein 5/Wnt receptor. CONCLUSIONS: The Wnt/ß-catenin signaling pathway components are functional and activated by mechanical loading in PDL cells. ß-catenin serves as an effector of mechanical signals in PDL cells.

TGFß-1 and Wnt-3a interact to induce unique gene expression profiles in murine embryonic palate mesenchymal cells.

Development of the secondary palate in mammals is a complex process under the control of numerous growth and differentiation factors that regulate key processes such as cell proliferation, synthesis of extracellular matrix molecules, and epithelial-mesenchymal transdifferentiation. Alterations in any one of these processes either through genetic mutation or environmental insult have the potential to lead to clefts of the secondary palate. Members of the TGFß family of cytokines are crucial mediators of these processes and emerging evidence supports a pivotal role for members of the Wnt family of secreted growth and differentiation factors. Previous work in this laboratory demonstrated cross-talk between the Wnt and TGFß signaling pathways in cultured mouse embryonic palate mesenchymal cells. In the current study we tested the hypothesis that unique gene expression profiles are induced in murine embryonic palate mesenchymal cells as a result of this cross-talk between the TGFß and Wnt signal transduction pathways.

The acceleration of implant osseointegration by liposomal Wnt3a.

The strength of a Wnt-based strategy for tissue regeneration lies in the central role that Wnts play in healing. Tissue injury triggers local Wnt activation at the site of damage, and this Wnt signal is required for the repair and/or regeneration of almost all tissues including bone, neural tissues, myocardium, and epidermis. We developed a biologically based approach to create a transient elevation in Wnt signaling in peri-implant tissues, and in doing so, accelerated bone formation around the implant. Our subsequent molecular and cellular analyses provide mechanistic insights into the basis for this pro-osteogenic effect. Given the essential role of Wnt signaling in bone formation, this protein-based approach may have widespread application in implant osseointegration.

Wnt proteins promote bone regeneration.

The Wnt signaling pathway plays a central role in bone development and homeostasis. In most cases, Wnt ligands promote bone growth, which has led to speculation that Wnt factors could be used to stimulate bone healing. We gained insights into the mechanism by which Wnt signaling regulates adult bone repair through the use of the mouse strain Axin2(LacZ/LacZ) in which the cellular response to Wnt is increased. We found that bone healing after injury is accelerated in Axin2(LacZ/LacZ) mice, a consequence of more robust proliferation and earlier differentiation of skeletal stem and progenitor cells. In parallel, we devised a biochemical strategy to increase the duration and strength of Wnt signaling at the sites of skeletal injury. Purified Wnt3a was packaged in liposomal vesicles and delivered to skeletal defects, where it stimulated the proliferation of skeletal progenitor cells and accelerated their differentiation into osteoblasts, cells responsible for bone growth. The end result was faster bone regeneration. Because Wnt signaling is conserved in mammalian tissue repair, this protein-based approach may have widespread applications in regenerative medicine.

Controlling the in vivo activity of Wnt liposomes.

Liposomes offer a method of delivering small molecules, nucleic acids, and proteins to sites within the body. Typically, bioactive materials are encapsulated within the liposomal aqueous core and liposomal phase transition is elicited by pH or temperature changes. We developed a new class of liposomes for the in vivo delivery of lipid-modified proteins. First, we show that the inclusion of a chromophore into the liposomal or vesosomal membrane renders these lipid vesicles extremely sensitive to very small (muJ) changes in energy. Next, we demonstrate that the lipid-modified Wnt protein is not encapsulated within a liposome but rather is tethered to the exoliposomal surface in an active configuration. When applied to intact skin, chromophore-modified liposomes do not penetrate past the corneal layer of the epidermis, but remain localized to the site of application. Injury to the epidermis allows rapid penetration of liposomes into the dermis, which suggests that mild forms of dermabrasion will greatly enhance transdermal delivery of liposome-packaged molecules. Finally, we demonstrate that topical application of Wnt3a liposomes rapidly stimulates proliferation of cells in the corneal layer, resulting in a thicker, more fibrillous epidermis.

Wnt signaling inhibits cementoblast differentiation and promotes proliferation.

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Liposomal packaging generates Wnt protein with in vivo biological activity.

Wnt signals exercise strong cell-biological and regenerative effects of considerable therapeutic value. There are, however, no specific Wnt agonists and no method for in vivo delivery of purified Wnt proteins. Wnts contain lipid adducts that are required for activity and we exploited this lipophilicity by packaging purified Wnt3a protein into lipid vesicles. Rather than being encapsulated, Wnts are tethered to the liposomal surface, where they enhance and sustain Wnt signaling in vitro. Molecules that effectively antagonize soluble Wnt3a protein but are ineffective against the Wnt3a signal presented by a cell in a paracrine or autocrine manner are also unable to block liposomal Wnt3a activity, suggesting that liposomal packaging mimics the biological state of active Wnts. When delivered subcutaneously, Wnt3a liposomes induce hair follicle neogenesis, demonstrating their robust biological activity in a regenerative context.