Structure-function analysis of secreted frizzled-related protein-1 for its Wnt antagonist function

Secreted frizzled-related proteins (sFRPs) are glycoproteins that are recognized as Wnt antagonists. To identify the functional domains that are involved in Wnt antagonist function, several sFRP-1 mutants and sFRP-1/sFRP-2 chimeras were generated. These mutants were characterized in an optimized T-cell factor (TCF)-luciferase based assay in U2OS human osteosarcoma cells. Deletions of the sFRP-1 cysteine rich domain (CRD) lead to the complete loss of Wnt antagonist function. A region between amino acids 73-86 within the second loop of the CRD of sFRP-1 was necessary for the optimal Wnt inhibitory function. Within this region, a conserved tyrosine residue played a critical role, and its change to neutral or polar amino acids lead to decreased Wnt inhibitory activity. The sFRP-1/sFRP-2 chimeras with the netrin domain of sFRP-1 replaced by corresponding sFRP-2 sequences showed 40-70% loss of Wnt antagonist function. The sFRP-1/sFRP-2 chimera with the replacement of C-terminal 19 amino acids of sFRP-1 with 11 amino acids of sFRP-2 resulted in 70% loss of activity indicating that carboxyl-terminal region of sFRP-1 is important for its Wnt inhibitory activity. The structure-function analysis studies of sFRP-1 clearly demonstrate the interaction of several functional domains for its optimal Wnt antagonist function.     

Secreted Frizzled-related proteins can regulate metanephric development

Wnt-4 signaling plays a critical role in kidney development and is associated with the epithelial conversion of the metanephric mesenchyme. Furthermore, secreted Frizzled-related proteins (sFRPs) that can bind Wnts are normally expressed in the developing metanephros, and function in other systems as modulators of Wnt signaling. sfrp-1 is distributed throughout the medullary and cortical stroma in the metanephros, but is absent from condensed mesenchyme and primitive tubular epithelia of the developing nephron where wnt-4 is highly expressed. In contrast, sfrp-2 is expressed in primitive tubules. To determine their role in kidney development, recombinant sFRP-1, sFRP-2 or combinations of both were applied to cultures of 13-dpc rat metanephroi. Both tubule formation and bud branching were markedly inhibited by sFRP-1, but concurrent sFRP-2 treatment restored some tubular differentiation and bud branching. sFRP-2 itself showed no effect on cultures of metanephroi. In cultures of isolated, induced rat metanephric mesenchymes, sFRP-1 blocked events associated with epithelial conversion (tubulogenesis and expression of lim-1, sfrp-2 and E-cadherin); however, it had no demonstrable effect on early events (compaction of mesenchyme and expression of wt1). As shown herein, sFRP-1 binds Wnt-4 with considerable avidity and inhibits the DNA-binding activity of TCF, an effector of Wnt signaling, while sFRP-2 had no effect on TCF activation. These observations suggest that sFRP-1 and sFRP-2 compete locally to regulate Wnt signaling during renal organogenesis. The antagonistic effect of sFRP-1 may be important either in preventing inappropriate development within differentiated areas of the medulla or in maintaining a population of cortical blastemal cells to facilitate further renal expansion. On the other hand, sFRP-2 might promote tubule formation by permitting Wnt-4 signaling in the presence of sFRP-1.     

The Wnt antagonist secreted frizzled-related protein-1 is a negative regulator of trabecular bone formation in adult mice

Previous studies have associated activation of canonical Wnt signaling in osteoblasts with elevated bone formation. Here we report that deletion of the murine Wnt antagonist, secreted frizzled-related protein (sFRP)-1, prolongs and enhances trabecular bone accrual in adult animals. sFRP-1 mRNA was expressed in bones and other tissues of +/+ mice but was not observed in -/- animals. Despite its broad tissue distribution, ablation of sFRP-1 did not affect blood and urine chemistries, most nonskeletal organs, or cortical bone. However, sFRP-1-/- mice exhibited increased trabecular bone mineral density, volume, and mineral apposition rate when compared with +/+ controls. The heightened trabecular bone mass of sFRP-1-/- mice was observed in adult animals between the ages of 13-52 wk, occurred in multiple skeletal sites, and was seen in both sexes. Mechanistically, loss of sFRP-1 reduced osteoblast and osteocyte apoptosis in vivo. In addition, deletion of sFRP-1 inhibited osteoblast lineage cell apoptosis while enhancing the proliferation and differentiation of these cells in vitro. Ablation of sFRP-1 also increased osteoclastogenesis in vitro, although changes in bone resorption were not observed in intact animals in vivo. Our findings demonstrate that deletion of sFRP-1 preferentially activates Wnt signaling in osteoblasts, leading to enhanced trabecular bone formation in adults.     

sFRP-1 binds via its netrin-related motif to the N-module of thrombospondin-1 and blocks thrombospondin-1 stimulation of MDA-MB-231 breast carcinoma cell adhesion and migration

Secreted frizzled-related protein (sFRP)-1 is a Wnt antagonist that inhibits breast carcinoma cell motility, whereas the secreted glycoprotein thrombospondin-1 stimulates adhesion and motility of the same cells. We examined whether thrombospondin-1 and sFRP-1 interact directly or indirectly to modulate cell behavior. Thrombospondin-1 bound sFRP-1 with an apparent K_d = 48 nM and the related sFRP-2 with a K_d = 95 nM. Thrombospondin-1 did not bind to the more distantly related sFRP-3. The association of thrombospondin-1 and sFRP-1 is primarily mediated by the amino-terminal N-module of thrombospondin-1 and the netrin domain of sFRP-1. sFRP-1 inhibited α3β1 integrin-mediated adhesion of MDA-MB-231 breast carcinoma cells to a surface coated with thrombospondin-1 or recombinant N-module, but not adhesion of the cells on immobilized fibronectin or type I collagen. sFRP-1 also inhibited thrombospondin-1-mediated migration of MDA-MB-231 and MDA-MB-468 breast carcinoma cells. Although sFRP-2 binds similarly to thrombospondin-1, it did not inhibit thrombospondin-1-stimulated adhesion. Thus, sFRP-1 binds to thrombospondin-1 and antagonizes stimulatory effects of thrombospondin-1 on breast carcinoma cell adhesion and motility. These results demonstrate that sFRP-1 can modulate breast cancer cell responses by interacting with thrombospondin-1 in addition to its known effects on Wnt signaling.     

Secreted frizzled-related protein-1 binds directly to Wingless and is a biphasic modulator of Wnt signaling

Secreted Frizzled-related protein-1 (sFRP-1) contains a cysteine-rich domain homologous to the putative Wnt-binding site of Frizzleds. To facilitate the biochemical and biological analysis of sFRP-1, we developed a mammalian recombinant expression system that yields approximately 3 mg of purified protein/liter of conditioned medium. Using this recombinant protein, we demonstrated that sFRP-1 and Wg (wingless) interact in enzyme-linked immunosorbent and co-precipitation assays. Surprisingly, a derivative lacking the cysteine-rich domain retained the ability to bind Wg. Cross-linking experiments performed with radioiodinated sFRP-1 provided definitive evidence that sFRP-1 and Wg bind directly to each other. Besides detecting a cross-linked complex consistent in size with 1:1 stoichiometry of sFRP-1 and Wg, we also observed a larger complex whose size suggested the presence of a second sFRP-1 molecule. The formation of both complexes was markedly enhanced by an optimal concentration of exogenous heparin, emphasizing the potential importance of heparan-sulfate proteoglycan in Wnt binding and signaling. sFRP-1 exerted a biphasic effect on Wg activity in an armadillo stabilization assay, increasing armadillo level at low concentrations but reducing it at higher concentrations. These results provide new insights about the Wnt binding and biological activity of sFRPs.     

The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-beta1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE(2) increased apoptosis threefold, while treatment of pre-osteocytes with TGF-beta1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival.     

Regulation of secreted Frizzled-related protein-1 by heparin

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.     

Place- and time-dependent expression of mouse sFRP-1 during development of the cerebral neocortex.

Wnts are a family of secreted proteins involved in multiple developmental mechanisms during nervous system development, including cell proliferation, cell migration, axon guidance and specification of cell positional information. We report here the expression of sFRP-1 mRNA, encoding a putative inhibitor of Wnt, in the developing mouse neocortex during the entire period when neurons for the neocortex are born. We show that sFRP-1 mRNA expression is spatially restricted to the proliferative zones during the period, when neurons are known to be generated in large numbers for the enlarging cortical plate.     

Secreted Wnt antagonists in scrub typhus

BackgroundThe mechanisms that control local and systemic inflammation in scrub typhus have only been partially elucidated. The wingless (Wnt) signaling pathways are emerging as important regulators of inflammation and infection, but have not been investigated in scrub typhus.Methodology/Principal findingsPlasma levels of secreted Wnt antagonists (i.e. DKK-1, sFRP-3, WIF-1 and SOST) were analyzed in patients with scrub typhus (n = 129), patients with similar febrile illness without O. tsutsugamushi infection (n = 31), febrile infectious disease controls, and in healthy controls (n = 31) from the same area of South India, and were correlated to markers of inflammation, immune and endothelial cell activation as well as for their association with organ specific dysfunction and mortality in these patients. We found i) Levels of SOST and in particular sFRP-3 and WIF-1 were markedly increased and DKK-1 decreased in scrub typhus patients at admission to the hospital compared to healthy controls. ii) In recovering scrub typhus patients, SOST, sFRP-3 and WIF-1 decreased and DKK-1 increased. iii) SOST was positively correlated with markers of monocyte/macrophage and endothelial/vascular activation as well as with renal dysfunction and poor outcome iv) Finally, regulation of Wnt pathways by O. tsutsugamushi in vitro in monocytes and ex vivo in mononuclear cells isolated from patients with scrub typhus, as evaluated by gene expression studies available in public repositories, revealed markedly attenuated canonical Wnt signaling.Conclusions/SignificanceOur findings suggest that scrub typhus is characterized by attenuated Wnt signaling possibly involving dysregulated levels of several secreted pathway antagonists. The secreted Wnt antagonist SOST was strongly associated with renal dysfunction and poor prognosis in these patients.     

Identification of a peptide binding motif for secreted frizzled-related protein-1

Secreted Frizzled-related proteins (sFRPs) bind Wnts and modulate their activity. To identify putative sFRP-1 binding motifs, we screened an M13 phage displayed combinatorial peptide library. A predominant motif, L/V-VDGRW-L/V, was present in approximately 70% of the phage that bound sFRP-1. Use of peptide/alkaline phosphatase chimeras and alanine scanning confirmed that the conserved motif was important for sFRP-1 recognition. The dissociation constant for a peptide/sFRP-1 complex was 3.9 microM. Additional analysis revealed that DGR was the core of the binding motif. Although Wnt proteins lack this sequence, other proteins possessing the DGR motif may function as novel binding partners for sFRP-1.     

Wnt signalling mediates the cross-talk between bone marrow derived pre-adipocytic and pre-osteoblastic cell populations

The mechanisms underlying the inverse relationship between osteogenic and adipogenic differentiation of bone marrow stromal cells (MSC) are not known in detail. We have previously established two cell lines from mouse bone marrow that are committed to either osteogenic (osteoblasts and chondrocytes) (mMSC^Bone) or adipogenic (mMSC^Adipo) lineage. To identify the molecular mechanism determining the lineage commitment, we compared the basal gene expression profile of mMSC^Bone versus mMSC^Adipo using Affymetrix GeneChip® MG430A 2.0 Array. Gene annotation analysis based on biological function revealed an over-representation of skeletal development genes in mMSC^Bone while genes related to lipid metabolism and immune response were highly expressed in mMSC^Adipo. In addition, there was a significant up-regulation of canonical Wnt signalling genes in mMSC^Bone compared to mMSC^Adipo (p < 0.006). Dual-luciferase assay and expression analysis of genes related to Wnt signalling demonstrated significant activation of Wnt signalling pathway in mMSC^Bone compared to mMSC^Adipo. Reduced Wnt activity in mMSC^Adipo was associated with increased expression of the Wnt inhibitor, secreted frizzled-related protein 1 (sFRP-1) at both mRNA and protein levels in mMSC^Adipo. Interestingly, conditioned medium (CM) collected from mMSC^Adipo (mMSC-CM^Adipo) inhibited osteoblast differentiation of mMSC, while depletion of sFRP-1 protein from mMSC-CM^Adipo abolished its inhibitory effect on osteoblast differentiation. Furthermore, treatment of mMSC with recombinant sFRP-1 resulted in a dose-dependent inhibition of osteoblast and stimulation of adipocyte differentiation. In conclusion, cross-talk exists between different populations of MSC in the bone marrow, and Wnt signalling functions as a molecular switch that determines the balance between osteoblastogenesis and adipogenesis.     

Regulated expression of sFRP-1 protein by the GeneSwitch system

The GeneSwitch system is a mifepristone-inducible expression system that provides exceptionally low uninduced and high-induced protein expression in mammalian cells. We have developed an adenovirus recombinant containing GeneSwitch protein driven by the GAL4-tk promoter, as well as recombinants containing sFRP-1 and luciferase reporter under the control of the GAL4-E1b promoter. Luciferase activity in A549 cells infected with the GeneSwitch and Luciferase viruses is very low in ethanol-treated cells, while the level of luciferase activity increases 200-fold in cells treated with mifepristone. Conditional expression of functional sFRP-1 is demonstrated in A549, human osteoblast, and CHO cell lines by either the co-infection of cells with sFRP-1 and GeneSwitch viruses or the infection of GeneSwitch expressing cell lines with sFRP-1 virus and subsequent treatment with mifepristone. The expression of sFRP-1 is seen as early as 4 h post-mifepristone treatment, reaching the highest levels at 20 h. The sFRP-1 protein is present in conditioned media, and the protein is functional based upon its ability to inhibit the Wnt-mediated activation of TCF-Luciferase reporter activity. The regulated expression of sFRP-1 utilizing adenovirus vectors provides an opportunity to address the contribution of sFRP-1 in the regulation of stem cell differentiation, maturation, and their function by modulating the Wnt signaling.     

Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts

Secreted frizzled-related proteins (sFRPs) are modulators of Wnt signaling. This study was undertaken for definitive assessment of contribution of different sFRPs in osteoblastic differentiation of mesenchymal progenitor cells and apoptosis of osteoblasts. Treatment of C3H10T1/2 cells with sFRP-2 at concentrations of 10, 50, and 100 nM and sFRP-4 at low concentrations (5 nM) significantly increased Wnt-3A-induced alkaline phosphatase (ALP) activities, whereas sFRP-1 or 3 did not. Retroviral transduction of the sFRP-2 but not other sFRPs also significantly enhanced ALP activity induced by β-glycerophosphate and ascorbic acid. Furthermore, transfection of all the sFRP expression vectors significantly increased β-catenin/TCF reporter activity and the effects were most prominent with sFRP-2 and -4. In osteoblast apoptosis assay, only sFRP-3 increased etoposide-induced apoptosis in MC3T3-E1 mouse osteoblasts. In conclusion, we found that different repertoires of sFRPs exert differential effects on osteoblastic differentiation of mouse mesenchymal cells and cellular apoptosis of mouse osteoblasts in vitro.     

Identification of iminooxothiazolidines as secreted frizzled related protein-1 inhibitors

Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure–activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.     

Lipid-independent secretion of a Drosophila Wnt protein

Wnt proteins comprise a large class of secreted signaling molecules with key roles during embryonic development and throughout adult life. Recently, much effort has been focused on understanding the factors that regulate Wnt signal production. For example, Porcupine and Wntless/Evi/Sprinter have been identified as being required in Wnt-producing cells for the processing and secretion of many Wnt proteins. Interestingly, in this study we find that WntD, a recently characterized Drosophila Wnt family member, does not require Porcupine or Wntless/Evi/Sprinter for its secretion or signaling activity. Because Porcupine is involved in post-translational lipid modification of Wnt proteins, we used a novel labeling method and mass spectrometry to ask whether WntD undergoes lipid modification and found that it does not. Although lipid modification is also hypothesized to be required for Wnt secretion, we find that WntD is secreted very efficiently. WntD secretion does, however, maintain a requirement for the secretory pathway component Rab1. Our results show that not all Wnt family members require lipid modification, Porcupine, or Wntless/Evi/Sprinter for secretion and suggest that different modes of secretion may exist for different Wnt proteins.     

Leucine-rich amelogenin peptide induces osteogenesis by activation of the Wnt pathway

We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stem cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/β-catenin signaling is activated upon LRAP treatment, as evidenced by elevated β-catenin level and increased Wnt reporter gene activity. Furthermore, a specific Wnt inhibitor sFRP-1 completely blocks the LRAP-mediated Wnt signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Wnt agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Wnt signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Wnt agonists and antagonists.     

Heads or tails: Wnts and anterior-posterior patterning.

Cell-cell communication is critical during embryogenesis for organizing the vertebrate body plan. Members of the Wnt family of secreted signaling molecules possess axis-inducing and posteriorizing activity when overexpressed. Wnt signals are modulated extracellularly by a diverse group of secreted Wnt antagonists and cofactors. Recent work has revealed that inhibition of posteriorly localized Wnt signaling by anteriorly localized Wnt antagonists is critical for inducing the anterior structures, forebrain and heart, from neural ectoderm and mesoderm, respectively. This review centers on the role that Wnts and Wnt antagonists play in the patterning of the vertebrate anterior-posterior axis.     

A secreted splice variant of the Xenopus frizzled-4 receptor is a biphasic modulator of Wnt signalling

Background

Activation of the Wnt signalling cascade is primarily based on the interplay between Wnt ligands, their receptors and extracellular modulators. One prominent family of extracellular modulators is represented by the SFRP (secreted Frizzled-related protein) family. These proteins have significant similarity to the extracellular domain of Frizzled receptors, suggesting that they bind Wnt ligands and inhibit signalling. The SFRP-type protein Fz4-v1, a splice variant of the Frizzled-4 receptor found in humans and Xenopus, was shown to augment Wnt/β-catenin signalling, and also interacts with those Wnt ligands that act on β-catenin-independent Wnt pathways.

Findings

Here we show that Xenopus Fz4-v1 can activate and inhibit the β-catenin-dependent Wnt pathway. Gain-of-function experiments revealed that high Wnt/β-catenin activity is inhibited by low and high concentrations of Fz4-v1. In contrast, signals generated by low amounts of Wnt ligands were enhanced by low concentrations of Fz4-v1 but were repressed by high concentrations. This biphasic activity of Fz4-v1 was not observed in non-canonical Wnt signalling. Fz4-v1 enhanced β-catenin-independent Wnt signalling triggered by either low or high doses of Wnt11. Antisense morpholino-mediated knock-down experiments demonstrated that in early Xenopus embryos Fz4-v1 is required for the migration of cranial neural crest cells and for the development of the dorsal fin.

Conclusions

For the first time, we show that a splice variant of the Frizzled-4 receptor modulates Wnt signalling in a dose-dependent, biphasic manner. These results also demonstrate that the cystein-rich domain (CRD), which is shared by Fz4-v1 and SFRPs, is sufficient for the biphasic activity of these secreted Wnt modulators.

     

Wnts differentially regulate colony growth and differentiation of chondrogenic rat calvaria cells

The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20-40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved.