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.     

Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation

Wnt signaling regulates a variety of developmental processes in animals. Although the beta-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here, we delineate a pathway in which Wnt3a signals through the Galpha(q/11) subunits of G proteins to activate phosphatidylinositol signaling and PKCdelta in the murine ST2 cells. Galpha(q/11)-PKCdelta signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCdelta homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCdelta-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation.     

Lymphotoxin-alpha 1 beta 2 and LIGHT induce classical and noncanonical NF-kappa B-dependent proinflammatory gene expression in vascular endothelial cells

Activation of the classical and noncanonical NF-kappaB pathways by ligation of the lymphotoxin (LT)-beta receptor (LTbetaR) plays a crucial role in lymphoid organogenesis and in the generation of ectopic lymphoid tissue at sites of chronic inflammation. Within these microenvironments, LTbetaR signaling regulates the phenotype of the specialized high endothelial cells. However, the direct effects of LTbetaR ligation on endothelial cells remain unclear. We therefore questioned whether LTbetaR ligation could directly activate endothelial cells and regulate classical and noncanonical NF-kappaB-dependent gene expression. We demonstrate that the LTbetaR ligands LIGHT and LTalpha1beta2 activate both NF-kappaB pathways in HUVECs and human dermal microvascular endothelial cells (HDMEC). Classical pathway activation was less robust than TNF-induced signaling; however, only LIGHT and LTalpha1beta2 and not TNF activated the noncanonical pathway. LIGHT and LTalpha1beta2 induced the expression of classical NF-kappaB-dependent genes in HUVEC, including those encoding the adhesion molecules E-selectin, ICAM-1, and VCAM-1. Consistent with this stimulation, LTbetaR ligation up-regulated T cell adhesion to HUVEC. Furthermore, the homeostatic chemokine CXCL12 was up-regulated by LIGHT and LTalpha1beta2 but not TNF in both HUVEC and HDMEC. Using HUVEC retrovirally transduced with dominant negative IkappaB kinase alpha, we demonstrate that CXCL12 expression is regulated by the noncanonical pathway in endothelial cells. Our findings therefore demonstrate that LTbetaR ligation regulates gene expression in endothelial cells via both NF-kappaB pathways and we identify CXCL12 as a bona fide noncanonical NF-kappaB-regulated gene in these cells.     

Bone cells crosstalk: noncanonical Roring in the Wnt

It is well established that canonical Wnt signaling in bone regulates bone mass. Much less is known about the?role of noncanonical Wnt signaling. Maeda and colleagues now report in Nature Medicine that Wnt5a-Ror2 crosstalk between bone cells enhances bone resorption, thereby negatively regulating skeletal homeostasis (Maeda et?al., 2012).     

Wnt5a signaling induces proliferation and survival of endothelial cells in vitro and expression of MMP-1 and Tie-2

Wnts are lipid-modified secreted glycoproteins that regulate diverse biological processes. We report that Wnt5a, which functions in noncanonical Wnt signaling, has activity on endothelial cells. Wnt5a is endogenously expressed in human primary endothelial cells and is expressed in murine vasculature at several sites in mouse embryos and tissues. Expression of exogenous Wnt5a in human endothelial cells promoted angiogenesis. Wnt5a induced noncanonical Wnt signaling in endothelial cells, as measured by Dishevelled and ERK1/2 phosphorylation, and inhibition of canonical Wnt signaling, a known property of Wnt5a. Wnt5a induced endothelial cell proliferation and enhanced cell survival under serum-deprived conditions. The Wnt5a-mediated proliferation was blocked by Frizzled-4 extracellular domain. Wnt5a expression enhanced capillary-like network formation, whereas reduction of Wnt5a expression decreased network formation. Reduced Wnt5a expression inhibited endothelial cell migration. Screening for Wnt5a-regulated genes in cultured endothelial cells identified several encoding angiogenic regulators, including matrix metalloproteinase-1, an interstitial collagenase, and Tie-2, a receptor for angiopoietins. Thus, Wnt5a acts through noncanonical Wnt signaling to promote angiogenesis.     

Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche

Wnt signaling is involved in self-renewal and maintenance of hematopoietic stem cells (HSCs); however, the particular role of noncanonical Wnt signaling in regulating HSCs in vivo is largely unknown. Here, we show Flamingo (Fmi) and Frizzled (Fz) 8, members of noncanonical Wnt signaling, both express in and functionally maintain quiescent long-term HSCs. Fmi regulates Fz8 distribution at the interface between HSCs and N-cadherin(+) osteoblasts (N-cad(+)OBs that enrich osteoprogenitors) in the niche. We further found that N-cad(+)OBs predominantly express noncanonical Wnt ligands and inhibitors of canonical Wnt signaling under homeostasis. Under stress, noncanonical Wnt signaling is attenuated and canonical Wnt signaling is enhanced in activation of HSCs. Mechanistically, noncanonical Wnt signaling mediated by Fz8 suppresses the Ca(2+)-NFAT- IFNγ pathway, directly or indirectly through the CDC42-CK1α complex and also antagonizes canonical Wnt signaling in HSCs. Taken together, our findings demonstrate that noncanonical Wnt signaling maintains quiescent long-term HSCs through Fmi and Fz8 interaction in the niche.     

Expression profiles of Wnt genes during neural differentiation of mouse embryonic stem cells

The Wnt family of secreted signaling proteins regulates many aspects of animal development and the behavior of several types of stem cells, including embryonic stem (ES) cells. Activation of canonical Wnt signaling has been shown to either inhibit or promote the differentiation of ES cells into neurons, depending on the stage of differentiation. Here, we describe the expression of all 19 mouse Wnt genes during this process. Using the well-established retinoic acid induction protocol we found that all Wnt genes except Wnt8b are expressed as ES cells differentiate into neurons, many of them in dynamic patterns. The expression pattern of 12 Wnt genes was analyzed quantitatively at 2-day intervals throughout neural differentiation, showing that multiple Wnt genes are expressed at each stage. A large proportion of these, including both canonical and noncanonical Wnts, are expressed at highest levels during later stages of differentiation. The complexity of the patterns observed indicates that disentangling specific roles for individual Wnt genes in the differentiation process will be a significant challenge.     

Zebrafish Rho kinase 2 acts downstream of Wnt11 to mediate cell polarity and effective convergence and extension movements

BACKGROUND: During vertebrate gastrulation convergence and extension (CE), movements narrow and lengthen embryonic tissues. In Xenopus and zebrafish, a noncanonical Wnt signaling pathway constitutes the vertebrate counterpart to the Drosophila planar cell polarity pathway and regulates mediolateral cell polarization underlying CE. Despite the identification of several signaling molecules required for normal CE, the downstream transducers regulating individual cell behaviors driving CE are only beginning to be elucidated. Moreover, how defective mediolateral cell polarity impacts CE is not understood.RESULTS: Here, we show that overexpression of zebrafish dominant-negative Rho kinase 2 (dnRok2) disrupts CE without altering cell fates, phenocopying noncanonical Wnt signaling mutants. Moreover, Rho kinase 2 (Rok2) overexpression partially suppresses the slb/wnt11 gastrulation phenotype, and ectopic expression of noncanonical Wnts modulates Rok2 intracellular distribution. In addition, time-lapse analyses associate defective dorsal convergence movements with impaired cell elongation, mediolateral orientation, and consequently failure to migrate along straight paths. Transplantation experiments reveal that dnRok2 cells in wild-type hosts neither elongate nor orient their axes. In contrast, wild-type cells are able to elongate their cell bodies in dnRok2 hosts, even though they fail to orient their axes.CONCLUSIONS: During zebrafish gastrulation, Rok2 acts downstream of noncanonical Wnt11 signaling to mediate mediolateral cell elongation required for dorsal cell movement along straight paths. Furthermore, elongation and orientation of the cell body are independent properties that require both cell-autonomous and nonautonomous Rok2 function.     

Wnts and wing: Wnt signaling in vertebrate limb development and musculoskeletal morphogenesis

In the past twenty years, secreted signaling molecules of the Wnt family have been found to play a central role in controlling embryonic development from hydra to human. In the developing vertebrate limb, Wnt signaling is required for limb bud initiation, early limb patterning (which is governed by several well-characterized signaling centers), and, finally, late limb morphogenesis events. Wnt ligands are unique, in that they can activate several different receptor-mediated signal transduction pathways. The most extensively studied Wnt pathway is the canonical Wnt pathway, which controls gene expression by stabilizing beta-catenin in regulating a diverse array of biological processes. Recently, more attention has been given to the noncanonical Wnt pathway, which is beta-catenin-independent. The noncanonical Wnt pathway signals through activating Ca(2+) flux, JNK activation, and both small and heterotrimeric G proteins, to induce changes in gene expression, cell adhesion, migration, and polarity. Abnormal Wnt signaling leads to developmental defects and human diseases affecting either tissue development or homeostasis. Further understanding of the biological function and signaling mechanism of Wnt signaling is essential for the development of novel preventive and therapeutic approaches of human diseases. This review provides a critical perspective on how Wnt signaling regulates different developmental processes. As Wnt signaling in tumor formation has been reviewed extensively elsewhere, this part is not included in the review of the clinical significance of Wnt signaling.     

A stromal cell-specific monoclonal antibody augments the stromal cell-dependent B lymphopoiesis.

We produced the mAb R25 against the stromal cell line ST2, which could support B lymphopoiesis in vitro. R25 enhanced the ability of ST2 to support B lymphopoiesis and precipitated molecules of 110 and 120 kDa (p110/120) from ST2 cell lysates. p110/120 were also expressed on other stromal and fibroblast cell lines but not on freshly isolated bone marrow hematopoietic cells, spleen cells, and lymphoid cell lines. However, R25 had no or weak effect on the stromal cell-dependent myelopoiesis. Even under conditions in which bone marrow cells were separated from stromal cells with a membrane filter, R25 could augment the stromal cell-dependent B lymphopoiesis. However, R25 did not induce the increase of IL-7 mRNA of ST2 cells. Taken together, these findings suggest that the stromal cell surface molecules p110/120 are involved in the stromal cell-dependent B lymphopoiesis and that certain soluble factors distinct from IL-7 may contribute to the p110/120-mediated B cell generation.     

The IKKα-dependent NF-κB p52/RelB noncanonical pathway is essential to sustain a CXCL12 autocrine loop in cells migrating in response to HMGB1

HMGB1 is a chromatin architectural protein that is released by dead or damaged cells at sites of tissue injury. Extracellular HMGB1 functions as a proinflammatory cytokine and chemoattractant for immune effector and progenitor cells. Previously, we have shown that the inhibitor of NF-κB kinase (IKK)β- and IKKα-dependent NF-κB signaling pathways are simultaneously required for cell migration to HMGB1. The IKKβ-dependent canonical pathway is needed to maintain expression of receptor for advanced glycation end products, the ubiquitously expressed receptor for HMGB1, but the target of the IKKα non-canonical pathway was not known. In this study, we show that the IKKα-dependent p52/RelB noncanonical pathway is critical to sustain CXCL12/SDF1 production in order for cells to migrate toward HMGB1. Using both mouse bone marrow-derived macrophages and mouse embryo fibroblasts (MEFs), it was observed that neutralization of CXCL12 by a CXCL12 mAb completely eliminated chemotaxis to HMGB1. In addition, the HMGB1 migration defect of IKKα KO and p52 KO cells could be rescued by adding recombinant CXCL12 to cells. Moreover, p52 KO MEFs stably transduced with a GFP retroviral vector that enforces physiologic expression of CXCL12 also showed near normal migration toward HMGB1. Finally, both AMD3100, a specific antagonist of CXCL12's G protein-coupled receptor CXCR4, and an anti-CXCR4 Ab blocked HMGB1 chemotactic responses. These results indicate that HMGB1-CXCL12 interplay drives cell migration toward HMGB1 by engaging receptors of both chemoattractants. This novel requirement for a second receptor-ligand pair enhances our understanding of the molecular mechanisms regulating HMGB1-dependent cell recruitment to sites of tissue injury.     

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance of noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.     

ZAP-70 Promotes the Infiltration of Malignant B-Lymphocytes into the Bone Marrow by Enhancing Signaling and Migration after CXCR4 Stimulation

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.     

Secreted frizzled-related protein 4 regulates two Wnt7a signaling pathways and inhibits proliferation in endometrial cancer cells

In the endometrium, hormonal effects on epithelial cells are often elicited through stromal hormone receptors via unknown paracrine mechanisms. Several lines of evidence support the hypothesis that Wnts participate in stromal-epithelial cell communication. Wnt7a is expressed in the luminal epithelium, whereas the extracellular modulator of Wnt signaling, secreted frizzled-related protein 4 (SFRP4), is localized to the stroma. Studies have reported that SFRP4 expression is significantly decreased in endometrial carcinoma and that both SFRP4 and Wnt7a genes are differentially regulated in response to estrogenic stimuli. Aberrant Wnt7a signaling irrevocably causes organ defects and infertility and contributes to the onset of disease. However, specific frizzled receptors (Fzd) that bind Wnt7a and the particular signal transduction pathway each Wnt7a-Fzd pair activates have not been identified. Additionally, the function of SFRP4 in the endometrium has not been addressed. We show here that Wnt7a coimmunoprecipitates with Fzd5, Fzd10, and SFRP4 in Ishikawa cells. Wnt7a binding to Fzd5 was shown to activate beta-catenin/canonical Wnt signaling and increase cellular proliferation. Conversely, Wnt7a signaling mediated by Fzd10 induced a noncanonical c-Jun NH2-terminal kinase-responsive pathway. SFRP4 suppresses activation of Wnt7a signaling in both an autocrine and paracrine manner. Stable overexpression of SFRP4 and treatment with recombinant SFRP4 protein inhibited endometrial cancer cell growth in vitro. These findings support a mechanism by which the nature of the Wnt7a signal in the endometrium is dependent on the Fzd repertoire of the cell and can be regulated by SFRP4.     

Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis

Despite their widespread expression, the in vivo recruitment activities of CCL19 (EBV-induced molecule 1 ligand chemokine) and CXCL12 (stromal cell-derived factor 1) have not been established. Furthermore, although CXCL13 (B lymphocyte chemoattractant) has been shown to induce lymphoid neogenesis through induction of lymphotoxin (LT)alpha1beta2, it is unclear whether other homeostatic chemokines have this property. In this work we show that ectopic expression in pancreatic islets of CCL19 leads to small infiltrates composed of lymphocytes and dendritic cells and containing high endothelial venules and stromal cells. Ectopic CXCL12 induced small infiltrates containing few T cells but enriched in dendritic cells, B cells, and plasma cells. Comparison of CCL19 transgenic mice with mice expressing CCL21 (secondary lymphoid tissue chemokine) revealed that CCL21 induced larger and more organized infiltrates. A more significant role for CCL21 is also suggested in lymphoid tissues, as CCL21 protein was found to be present in lymph nodes and spleen at much higher concentrations than CCL19. CCL19 and CCL21 but not CXCL12 induced LTalpha1beta2 expression on naive CD4 T cells, and treatment of CCL21 transgenic mice with LTbetaR-Fc antagonized development of organized lymphoid structures. LTalpha1beta2 was also induced on naive T cells by the cytokines IL-4 and IL-7. These studies establish that CCL19 and CXCL12 are sufficient to mediate cell recruitment in vivo and they indicate that LTalpha1beta2 may function downstream of CCL21, CCL19, and IL-2 family cytokines in normal and pathological lymphoid tissue development.     

Inhibition of canonical WNT signaling pathway by β-catenin/CBP inhibitor ICG-001 ameliorates liver fibrosis in vivo through suppression of stromal CXCL12

Quiescent hepatic stellate cells (HSCs), in response to liver injury, undergo characteristic morphological transformation into proliferative, contractile and ECM-producing myofibroblasts. In this study, we investigated the implication of canonical Wnt signaling pathway in HSCs and liver fibrogenesis. Canonical Wnt signaling pathway activation and inhibition using β-catenin/CBP inhibitor ICG001 was examined in-vitro in TGFβ-activated 3T3, LX2, primary human HSCs, and in-vivo in CCl₄-induced acute liver injury mouse model. Fibroblasts-conditioned medium studies were performed to assess the Wnt-regulated paracrine factors involved in crosstalk between HSCs-macrophages and HSCs-endothelial cells. Canonical Wnt signaling pathway components were significantly up-regulated in-vitro and in-vivo. In-vitro, ICG-001 significantly inhibited fibrotic parameters, 3D-collagen contractility and wound healing. Conditioned medium induced fibroblasts-mediated macrophage and endothelial cells activation was significantly inhibited by ICG-001. In-vivo, ICG-001 significantly attenuated collagen accumulation and HSC activation. Interestingly, ICG-001 drastically inhibited macrophage infiltration, intrahepatic inflammation and angiogenesis. We further analyzed the paracrine factors involved in Wnt-mediated effects and found CXCL12 was significantly suppressed both in-vitro and in-vivo following Wnt inhibition. Wnt-regulated CXCL12 secretion from activated HSCs potentiated macrophage infiltration and activation, and angiogenesis. Pharmacological inhibition of canonical Wnt signaling pathway via suppression of stromal CXCL12 suggests a potential therapeutic approach targeting activated HSCs in liver fibrosis.