Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis

The signaling molecule Wnt regulates bone homeostasis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Impairment of canonical Wnt signaling causes bone loss in arthritis and osteoporosis; however, it is unclear how noncanonical Wnt signaling regulates bone resorption. Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor (Ror) proteins. We showed that Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhanced osteoclastogenesis. Osteoblast-lineage cells expressed Wnt5a, whereas osteoclast precursors expressed Ror2. Mice deficient in either Wnt5a or Ror2, and those with either osteoclast precursor-specific Ror2 deficiency or osteoblast-lineage cell-specific Wnt5a deficiency showed impaired osteoclastogenesis. Wnt5a-Ror2 signals enhanced receptor activator of nuclear factor-κB (RANK) expression in osteoclast precursors by activating JNK and recruiting c-Jun on the promoter of the gene encoding RANK, thereby enhancing RANK ligand (RANKL)-induced osteoclastogenesis. A soluble form of Ror2 acted as a decoy receptor of Wnt5a and abrogated bone destruction in mouse arthritis models. Our results suggest that the Wnt5a-Ror2 pathway is crucial for osteoclastogenesis in physiological and pathological environments and represents a therapeutic target for bone diseases, including arthritis.     

Rab35 is required for Wnt5a/Dvl2-induced Rac1 activation and cell migration in MCF-7 breast cancer cells

The small GTPases regulate many major biological processes in both tumorigenesis and tumor progression such as cell survival, actin cytoskeleton organization, cell polarity and movement. Wnt5a, a non-canonical Wnt family member, is implicated in the activation of small GTPases in breast cancer. We previously demonstrated that Wnt5a signaling stimulates the migration of breast cancer cells MDA-MB-231 via activating RhoA. However, we found here that RhoA activation was not enhanced by Wnt5a in breast cancer cells MCF-7. The conflicting results prompted us to further probe novel small GTPases in response to Wnt5a and investigate the mechanisms whereby cell migration is regulated. We showed here that Wnt5a dose dependently activated Dvl2, Rab35 and Rac1 and subsequently promoted the migration of MCF-7 cells, which was, however, abolished by knocking down Wnt5a expression via small interfering RNA (siRNA) transfection. Dvl2 siRNA significantly decreased background and Wnt5a-induced Rab35/Rac1 activation and, consequently, cell migration. Rab35 short hairpin RNA (shRNA) remarkably inhibited background and Wnt5a-induced Rac1 activation and cell migration. Additionally, blockade of Rac1 activation with Rac1 siRNA suppressed background and Wnt5a-induced cell migration. Co-immunoprecipitation and immunofluorescence assays showed that Dvl2 bound to Rab35 in mammalian cells. Taken together, we demonstrated that Wnt5a promotes breast cancer cell migration via the Dvl2/Rab35/Rac1 signaling pathway. These findings implicate Wnt5a signaling in regulating small GTPases, which could be targeted for manipulating breast cancer cell migration.     

HECT Domain-containing E3 Ubiquitin Ligase NEDD4L Negatively Regulates Wnt Signaling by Targeting Dishevelled for Proteasomal Degradation

Wnt signaling plays a pivotal role in embryogenesis and tissue homeostasis. Dishevelled (Dvl) is a central mediator for both Wnt/β-catenin and Wnt/planar cell polarity pathways. NEDD4L, an E3 ubiquitin ligase, has been shown to regulate ion channel activity, cell signaling, and cell polarity. Here, we report a novel role of NEDD4L in the regulation of Wnt signaling. NEDD4L induces Dvl2 polyubiquitination and targets Dvl2 for proteasomal degradation. Interestingly, the NEDD4L-mediated ubiquitination of Dvl2 is Lys-6, Lys-27, and Lys-29 linked but not typical Lys-48-linked ubiquitination. Consistent with the role of Dvl in both Wnt/β-catenin and Wnt/planar cell polarity signaling, NEDD4L regulates the cellular β-catenin level and Rac1, RhoA, and JNK activities. We have further identified a hierarchical regulation that Wnt5a induces JNK-mediated phosphorylation of NEDD4L, which in turn promotes its ability to degrade Dvl2. Finally, we show that NEDD4L inhibits Dvl2-induced axis duplication in Xenopus embryos. Our work thus demonstrates that NEDD4L is a negative feedback regulator of Wnt signaling.     

Tiam1 Regulates the Wnt/Dvl/Rac1 Signaling Pathway and the Differentiation of Midbrain Dopaminergic Neurons

Understanding the mechanisms that drive the differentiation of dopaminergic (DA) neurons is crucial for successful development of novel therapies for Parkinson''s disease, in which DA neurons progressively degenerate. However, the mechanisms underlying the differentiation-promoting effects of Wnt5a on DA precursors are poorly understood. Here, we present the molecular and functional characterization of a signaling pathway downstream of Wnt5a, the Wnt/Dvl/Rac1 pathway. First, we characterize the interaction between Rac1 and Dvl and identify the N-terminal part of Dvl3 as necessary for Rac1 binding. Next, we show that Tiam1, a Rac1 guanosine exchange factor (GEF), is expressed in the ventral midbrain, interacts with Dvl, facilitates Dvl-Rac1 interaction, and is required for Dvl- or Wnt5a-induced activation of Rac1. Moreover, we show that Wnt5a promotes whereas casein kinase 1 (CK1), a negative regulator of the Wnt/Dvl/Rac1 pathway, abolishes the interactions between Dvl and Tiam1. Finally, using ventral midbrain neurosphere cultures, we demonstrate that the generation of DA neurons in culture is impaired after Tiam1 knockdown, indicating that Tiam1 is required for midbrain DA differentiation. In summary, our data identify Tiam1 as a novel regulator of DA neuron development and as a Dvl-associated and Rac1-specific GEF acting in the Wnt/Dvl/Rac1 pathway.     

Phosphorylated c-Jun and Fra-1 induce matrix metalloproteinase-1 and thereby regulate invasion activity of 143B osteosarcoma cells

Osteosarcoma is the most common primary malignancy of bone and patients often develop pulmonary metastases. Despite the advances in surgical and medical management, the mechanisms underlying human osteosarcoma progression and metastasis remain to be elucidated. Gene expression profiles were compared by the cDNA microarray technique between two different human osteosarcoma sublines, MNNG/HOS and 143B, which differ greatly in spontaneous pulmonary metastatic potential. Here we report an enhanced expression of matrix metalloproteinase (MMP)-1 in the highly metastatic human osteosarcoma cell line 143B. Moreover, the in vitro invasion activity of 143B cells was MMP-1-dependent. The activator protein (AP)-1 binding site in the MMP-1 gene promoter was required for the constitutive expression of MMP-1 in 143B cells. Two AP-1 components, c-Jun and Fra-1, were phosphorylated, and bound to the AP-1 binding site of the MMP-1 promoter in 143B cells. Activated c-Jun and Fra-1 were essential for MMP-1 gene expression in 143B cells. Mitogen-activated protein kinase pathways including the c-Jun NH₂-terminal kinase and the extracellular signal-regulated kinase activate c-Jun and Fra-1 and thereby regulate c-Jun/Fra-1 mediated events, establishing the mitogen-activated protein kinase/AP-1/MMP-1 axis as important in 143B cells. These data suggest that MMP-1 plays a central role in osteosarcoma invasion. Accordingly, MMP-1 might be a biomarker and therapeutic target for invasive osteosarcomas and pulmonary metastases.     

Atypical Protein Kinase Cι Is Required for Wnt3a-dependent Neurite Outgrowth and Binds to Phosphorylated Dishevelled 2

Previously we reported that Wnt3a-dependent neurite outgrowth in Ewing sarcoma family tumor cell lines was mediated by Frizzled3, Dishevelled (Dvl), and c-Jun N-terminal kinase (Endo, Y., Beauchamp, E., Woods, D., Taylor, W. G., Toretsky, J. A., Uren, A., and Rubin, J. S. (2008) Mol. Cell. Biol. 28, 2368–2379). Subsequently, we observed that Dvl2/3 phosphorylation correlated with neurite outgrowth and that casein kinase 1δ, one of the enzymes that mediate Wnt3a-dependent Dvl phosphorylation, was required for neurite extension (Greer, Y. E., and Rubin, J. S. (2011) J. Cell Biol. 192, 993–1004). However, the functional relevance of Dvl phosphorylation in neurite outgrowth was not established. Dvl1 has been shown by others to be important for axon specification in hippocampal neurons via an interaction with atypical PKCζ, but the role of Dvl phosphorylation was not evaluated. Here we report that Ewing sarcoma family tumor cells express PKCι but not PKCζ. Wnt3a stimulated PKCι activation and caused a punctate distribution of pPKCι in the neurites and cytoplasm, with a particularly intense signal at the centrosome. Knockdown of PKCι expression with siRNA reagents blocked neurite formation in response to Wnt3a. Aurothiomalate, a specific inhibitor of PKCι/Par6 binding, also suppressed neurite extension. Wnt3a enhanced the co-immunoprecipitation of endogenous PKCι and Dvl2. Although FLAG-tagged wild-type Dvl2 immunoprecipitated with PKCι, a phosphorylation-deficient Dvl2 derivative did not. This derivative also was unable to rescue neurite outgrowth when endogenous Dvl2/3 was suppressed by siRNA (González-Sancho, J. M., Greer, Y. E., Abrahams, C. L., Takigawa, Y., Baljinnyam, B., Lee, K. H., Lee, K. S., Rubin, J. S., and Brown, A. M. (2013) J. Biol. Chem. 288, 9428–9437). Taken together, these results suggest that site-specific Dvl2 phosphorylation is required for Dvl2 association with PKCι. This interaction is likely to be one of the mechanisms essential for Wnt3a-dependent neurite outgrowth.     

Ror2/Frizzled Complex Mediates Wnt5a-Induced AP-1 Activation by Regulating Dishevelled Polymerization

The receptor tyrosine kinase Ror2 acts as a receptor or coreceptor for Wnt5a to mediate Wnt5a-induced activation of the Wnt/JNK pathway and inhibition of the β-catenin-dependent canonical Wnt pathway. However, little is known about how Ror2 cooperates with another receptor component(s) to mediate Wnt5a signaling. We show here that Ror2 regulates Wnt5a-induced polymerization of Dishevelled (Dvl) and that this Ror2-mediated regulation of Dvl is independent of the cytoplasmic region of Ror2. Ror2 can associate with Frizzled7 (Fz7) via its extracellular cysteine-rich domain to form a receptor complex that is required for the regulation of Dvl and activation of the AP-1 promoter after Wnt5a stimulation. Suppressed expression of Fz7 indeed results in the inhibition of Wnt5a-induced polymerization of Dvl and AP-1 activation. Interestingly, both the DIX and the DEP domains of Dvl are indispensable for Dvl polymerization and subsequent AP-1 activation after Wnt5a stimulation. We further show that polymerized Dvl is colocalized with Rac1 and that suppressed expression of Rac1 inhibits Wnt5a-induced AP-1 activation. Collectively, our results indicate that Ror2/Fz receptor complex plays an important role in the Wnt5a/Rac1/AP-1 pathway by regulating the polymerization of Dvl.Wnt proteins can elicit β-catenin-dependent and -independent signaling pathways (2, 20, 46). Ror2 is a member of the Ror family of receptor tyrosine kinases and plays essential roles in developmental morphogenesis (21, 26, 31, 32, 44). Ror2 has been shown to act as a receptor or coreceptor for Wnt5a to activate the β-catenin-independent signaling pathway, involving JNK/c-Jun (AP-1), Src and Ca²⁺, which are essential for cell polarity, migration, and cancer cell invasion (8, 14, 28-31, 37). Wnt5a/Ror2 signaling also plays a crucial role in inhibiting the β-catenin-dependent signaling pathway (25). Structure-function analyses of Ror2 revealed that Ror2 mediates Wnt5a signaling through distinct mechanisms dependent on and independent of its kinase activity, i.e., Wnt5a-induced migration of fibroblast cells requires the cytoplasmic C-terminal portion of Ror2 but not its intrinsic kinase activity (28), whereas the intrinsic kinase activity of Ror2 is indispensable for extracellular matrix (ECM) degradation of osteosarcoma cells (8). In addition, inhibition of the β-catenin-dependent signaling pathway by Wnt5a also requires the intrinsic kinase activity of Ror2 (24). Importantly, the Caenorhabditis elegans ortholog of Ror2, CAM-1, also has the kinase activity-dependent and -independent functions (9, 12, 13). Furthermore, CAM-1 exhibits the cytoplasmic region-independent functions, including cell migration (17), synaptic transmission at the neuromuscular junction (10), and inhibition of the β-catenin-dependent signaling pathway (11), although their underlying molecular mechanisms remain to be determined. However, it is unknown whether or not Ror2 also exhibits the cytoplasmic region-independent functions in other organisms.Dishevelled (Dvl) is an essential mediator of both the β-catenin-dependent and -independent signaling pathways. We have previously reported that both Ror2 and Dvl are required for Wnt5a-induced cell migration (28). However, the relationship between Ror2 and Dvl in Wnt5a signaling remains unclear. It has been reported that Dvl has an ability to form dynamic polymers, which are crucial for activating the β-catenin-dependent signaling pathway probably by serving as a scaffold for Axin recruitment (39, 41). However, there is no direct evidence showing that Wnt stimulation indeed induces dynamic formation of Dvl polymers. In addition, it remains unclear whether or not the polymerization of Dvl is involved in the β-catenin-independent signaling pathway.In the present study we show that Wnt5a induces dynamic polymerization of Dvl2 via a receptor complex containing both Ror2 and Frizzled (Fz)7, even in the absence of the cytoplasmic region of Ror2. We further provide evidence indicating that Ror2/Fz7 receptor complex plays an important role in Wnt5a/Rac1/AP-1 pathway by regulating polymerization of Dvl2.     

Disheveled mediated planar cell polarity signaling is required in the second heart field lineage for outflow tract morphogenesis

Disheveled (Dvl) is a key regulator of both the canonical Wnt and the planar cell polarity (PCP) pathway. Previous genetic studies in mice indicated that outflow tract (OFT) formation requires Dvl1 and 2, but it was unclear which pathway was involved and whether Dvl1/2-mediated signaling was required in the second heart field (SHF) or the cardiac neural crest (CNC) lineage, both of which are critical for OFT development. In this study, we used Dvl1/2 null mice and a set of Dvl2 BAC transgenes that function in a pathway-specific fashion to demonstrate that Dvl1/2-mediated PCP signaling is essential for OFT formation. Lineage-specific gene-ablation further indicated that Dvl1/2 function is dispensable in the CNC, but required in the SHF for OFT lengthening to promote cardiac looping. Mutating the core PCP gene Vangl2 and non-canonical Wnt gene Wnt5a recapitulated the OFT morphogenesis defects observed in Dvl1/2 mutants. Consistent with genetic interaction studies suggesting that Wnt5a signals through the PCP pathway, Dvl1/2 and Wnt5a mutants display aberrant cell packing and defective actin polymerization and filopodia formation specifically in SHF cells in the caudal splanchnic mesoderm (SpM), where Wnt5a and Dvl2 are co-expressed specifically. Our results reveal a critical role of PCP signaling in the SHF during early OFT lengthening and cardiac looping and suggest that a Wnt5a→ Dvl PCP signaling cascade may regulate actin polymerization and protrusive cell behavior in the caudal SpM to promote SHF deployment, OFT lengthening and cardiac looping.     

Aberrant polycystin-1 expression results in modification of activator protein-1 activity, whereas Wnt signaling remains unaffected

Polycystin-1, the polycystic kidney disease 1 gene product, has been implicated in several signaling complexes that are known to regulate essential cellular functions. We investigated the role of polycystin-1 in Wnt signaling and activator protein-1 (AP-1) activation. To this aim, a membrane-targeted construct encoding the conserved C-terminal region of mouse polycystin-1 reported to mediate signal transduction activity was expressed in human embryonic and renal epithelial cells. To ensure specificity and minimal cotransfection effects, we focused our study on the endogenous proteins that actually transduce the signals, beta-catenin and T-cell factor/lymphoid-enhancing factor for Wnt signaling and (phosphorylated) c-Jun, ATF2, and c-Fos for AP-1. Our data indicate that the C-terminal region of polycystin-1 activates AP-1 by inducing phosphorylation and expression of at least c-Jun and ATF2, whereas c-Fos was not affected. Under our experimental conditions, polycystin-1 did not modulate Wnt signaling. AP-1 activity was aberrant in human autosomal dominant polycystic kidney disease (ADPKD) renal cystic epithelial cells and in renal epithelial cells expressing transgenic full-length polycystin-1, resulting in decreased Jun-ATF and increased Jun-Fos activity, whereas Wnt signaling remained unaffected. Since our data indicate that aberrant polycystin-1 expression results in altered AP-1 activity, polycystin-1 may be required for adequate AP-1 activity.     

Up-regulated lnc-SNHG1 contributes to osteosarcoma progression through sequestration of miR-577 and activation of WNT2B/Wnt/β-catenin pathway

Long noncoding RNA small nucleolar RNA host gene 1 (lnc-SNHG1) was reported to play an oncogenic role in the progression of cancers. However, the roles of SNHG1 and its molecular mechanism in osteosarcoma (OS) cells are largely unknown. In present study, we found that the expression of SNHG1 was up-regulated in OS tissues and cell lines. OS patients with the high SNHG1 expression were positively correlated with tumor size, TNM stage and lymph node metastasis. In addition, SNHG1 overexpression promoted cell proliferation, cell migration and EMT process in U2OS and MG63 cells and tumor growth in vivo. Furthermore, we also found that miR-577 could act as a ceRNAof SNHG1 in OS cells and the promotion of OS progression induced by lnc-SNHG1 overexpression required the inactivity of miR-577. Besides, we identified that WNT2B acted as a target of miR-577, and WNT2B played the oncogenic role in OS cells by activating Wnt/β-catenin pathway. In short, our study suggested that lnc-SNHG1 could promote OS progression via miR-577 and WNT2B. The lnc-SNHG1/miR-577/WNT2B/Wnt/β-catenin axis regulatory network might provide a potential new therapeutic strategy for OS treatment.     

Functional Consequences of Wnt-induced Dishevelled 2 Phosphorylation in Canonical and Noncanonical Wnt Signaling

Dishevelled (Dvl) proteins are intracellular effectors of Wnt signaling that have essential roles in both canonical and noncanonical Wnt pathways. It has long been known that Wnts stimulate Dvl phosphorylation, but relatively little is known about its functional significance. We have previously reported that both Wnt3a and Wnt5a induce Dvl2 phosphorylation that is associated with an electrophoretic mobility shift and loss of recognition by monoclonal antibody 10B5. In the present study, we mapped the 10B5 epitope to a 16-amino acid segment of human Dvl2 (residues 594–609) that contains four Ser/Thr residues. Alanine substitution of these residues (P4m) eliminated the mobility shift induced by either Wnt3a or Wnt5a. The Dvl2 P4m mutant showed a modest increase in canonical Wnt/β-catenin signaling activity relative to wild type. Consistent with this finding, Dvl2 4Pm preferentially localized to cytoplasmic puncta. In contrast to wild-type Dvl2, however, the P4m mutant was unable to rescue Wnt3a-dependent neurite outgrowth in TC-32 cells following suppression of endogenous Dvl2/3. Earlier work has implicated casein kinase 1δ/ϵ as responsible for the Dvl mobility shift, and a CK1δ in vitro kinase assay confirmed that Ser⁵⁹⁴, Thr⁵⁹⁵, and Ser⁵⁹⁷ of Dvl2 are CK1 targets. Alanine substitution of these three residues was sufficient to abrogate the Wnt-dependent mobility shift. Thus, we have identified a cluster of Ser/Thr residues in the C-terminal domain of Dvl2 that are Wnt-induced phosphorylation (WIP) sites. Our results indicate that phosphorylation at the WIP sites reduces Dvl accumulation in puncta and attenuates β-catenin signaling, whereas it enables noncanonical signaling that is required for neurite outgrowth.     

LRP5 Signaling in Osteosarcomagenesis: a Cautionary Tale of Translation from Cell Lines to Tumors

Previous reports document expression of low-density lipoprotein receptor-related protein 5 (LRP5) in osteosarcoma (OS) tissue. Expression of this Wnt receptor correlated with metastatic disease and poor disease-free survival. Forced expression of dominant-negative LRP5 (dnLRP5), which lacks the membrane binding domain of the native protein and therefore functions as a soluble receptor-sponge for Wnt ligands, reduced in vitro cellular invasion and in vivo xenograft tumor growth for osteosarcoma cell lines. Here, we use a genetically engineered mouse model of osteosarcomagenesis with and without expression of dnLRP5 to assess to what degree tumorigenesis is affected and whether Wnt/β-catenin signaling is circumvented or maintained. Each cohort of mice developed osteosarcoma at a similar ultimate prevalence, but after a slightly increased latency in those also expressing dnLRP5. On histology, there was no difference between groups, despite previous reports that the dnLRP5 osteosarcoma cells specifically undergo a mesenchymal-to-epithelial transition in vitro. Finally, immunohistochemistry showed the presence of cytosolic and nuclear β-catenin and nuclear Cyclin D1, markers consistent with preserved Wnt/β-catenin signaling despite constitutive blockade of the cell surface receipt of Wnt signaling ligand. These data suggest that canonical Wnt signaling plays a role in OS progression and that while blockade of singular nodes in signaling pathways can have dramatic effects on individual cell lines, real tumors readily evade such focused attacks.     

Transient overexpression of murine dishevelled genes results in apoptotic cell death

The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are implicated in Wnt signal transduction. In mammals, the manner in which Wnt signals are transduced remains unclear. The biochemical and molecular mechanisms defining the Wnt-1 pathway are of great interest because of its important role in development and its activation in murine breast tumors. In order to elucidate Dvl's role in Wnt signaling, we attempted to overexpress Dvl in cells, but were unable to obtain stable cell lines. We show here that the overexpression of Dvl genes alters nuclear and cellular morphology of COS-1 and C57MG cells and causes cell death due to the induction of apoptosis. Deletion studies demonstrate that all three conserved domains of Dvl (DIX, PDZ, and DEP) are required for Dvl-mediated cell death. Coexpression of protein phosphatase 2Calpha, a Dvl-interacting protein identified in yeast two-hybrid studies, protects cells from the cell death observed in cells overexpressing Dvl alone. Furthermore, the adenomatous polyposis coli (APC) gene product appears to be required for Dvl-mediated cell death. The relevance of these findings to Wnt signal transduction, as well as to developmental processes and disease, are discussed.