Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function

Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/β-catenin signaling. We found the extracellular β-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.     

Wnt antagonists bind through a short peptide to the first β-propeller domain of LRP5/6

The Wnt pathway inhibitors DKK1 and sclerostin (SOST) are important therapeutic targets in diseases involving bone loss or damage. It has been appreciated that Wnt coreceptors LRP5/6 are also important, as human missense mutations that result in bone overgrowth (bone mineral density, or BMD, mutations) cluster to the E1 propeller domain of LRP5. Here, we report a crystal structure of LRP6 E1 bound to an antibody, revealing that the E1 domain is a peptide recognition module. Remarkably, the consensus E1 binding sequence is a close match to a conserved tripeptide motif present in all Wnt inhibitors that bind LRP5/6. We show that this motif is important for DKK1 and SOST binding to LRP6 and for inhibitory function, providing a detailed structural explanation for the effect of the BMD mutations.     

Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6

LDL receptor-related proteins 5 and 6 (LRP5/6) are coreceptors for Wnt growth factors, and also bind Dkk proteins, secreted inhibitors of Wnt signaling. The LRP5/6 ectodomain contains four β-propeller/EGF-like domain repeats. The first two repeats, LRP6(1-2), bind to several Wnt variants, whereas LRP6(3-4) binds other Wnts. We present the crystal structure of the Dkk1 C-terminal domain bound to LRP6(3-4), and show that the Dkk1 N-terminal domain binds to LRP6(1-2), demonstrating that a single Dkk1 molecule can bind to both portions of the LRP6 ectodomain and thereby inhibit different Wnts. Small-angle X-ray scattering analysis of LRP6(1-4) bound to a noninhibitory antibody fragment or to full-length Dkk1 shows that in both cases the ectodomain adopts a curved conformation that places the first three repeats at a similar height relative to the membrane. Thus, Wnts bound to either portion of the LRP6 ectodomain likely bear a similar spatial relationship to Frizzled coreceptors.     

Mesd is a general inhibitor of different Wnt ligands in Wnt/LRP signaling and inhibits PC-3 tumor growth in vivo

Mesd is a specialized chaperone for Wnt co-receptor low-density lipoprotein receptor-related protein-5 (LRP5) and LRP6, which contain four β-propeller/epidermal growth factor modules, named E1 to E4 from N- to C-terminal, in their extracellular domains. Herein, we demonstrated that recombinant Mesd protein is a general Wnt inhibitor that blocks Wnt/β-catenin signaling induced not only by LRP6 E1-E2-binding Wnts but also by LRP6 E3-E4-binding Wnts. We also found that Mesd suppressed Wnt/β-catenin signaling induced by Wnt1 in prostate cancer PC-3 cells, and inhibited tumor growth in PC-3 xenograft model. Our results indicate that Mesd is a universal inhibitor of Wnt/LRP signaling on the cell surface.     

Reconstitution of a Frizzled8��Wnt3a��LRP6 Signaling Complex Reveals Multiple Wnt and Dkk1 Binding Sites on LRP6

Wnt/β-catenin signaling is initiated at the cell surface by association of secreted Wnt with its receptors Frizzled (Fz) and low density lipoprotein receptor-related protein 5/6 (LRP5/6). The study of these molecular interactions has been a significant technical challenge because the proteins have been inaccessible in sufficient purity and quantity. In this report we describe insect cell expression and purification of soluble mouse Fz8 cysteine-rich domain and human LRP6 extracellular domain and show that they inhibit Wnt/β-catenin signaling in cellular assays. We determine the binding affinities of Wnts and Dickkopf 1 (Dkk1) to the relevant co-receptors and reconstitute in vitro the Fz8 CRD·Wnt3a·LRP6 signaling complex. Using purified fragments of LRP6, we further show that Wnt3a binds to a region including only the third and fourth β-propeller domains of LRP6 (E3E4). Surprisingly, we find that Wnt9b binds to a different part of the LRP6 extracellular domain, E1E2, and we demonstrate that Wnt3a and Wnt9b can bind to LRP6 simultaneously. Dkk1 binds to both E1E2 and E3E4 fragments and competes with both Wnt3a and Wnt9b for binding to LRP6. The existence of multiple, independent Wnt binding sites on the LRP6 co-receptor suggests new possibilities for the architecture of Wnt signaling complexes and a model for broad-spectrum inhibition of Wnt/β-catenin signaling by Dkk1.     

Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling

The loss of the SOST gene product sclerostin leads to sclerosteosis characterized by high bone mass. In this report, we found that sclerostin could antagonize canonical Wnt signaling in human embryonic kidney A293T cells and mouse osteoblastic MC3T3 cells. This sclerostin-mediated antagonism could be reversed by overexpression of Wnt co-receptor low density lipoprotein receptor-related protein (LRP) 5. In addition, we found that sclerostin bound to LRP5 as well as LRP6 and identified the first two YWTD-EGF repeat domains of LRP5 as being responsible for the binding. Although these two repeat domains are required for transduction of canonical Wnt signals, canonical Wnt did not appear to compete with sclerostin for binding to LRP5. Examination of the expression of sclerostin and Wnt7b, an autocrine canonical Wnt, during primary calvarial osteoblast differentiation revealed that sclerostin is expressed at late stages of osteoblast differentiation coinciding with the expression of osteogenic marker osteocalcin and trailing after the expression of Wnt7b. Given the plethora of evidence indicating that canonical Wnt signaling stimulates osteogenesis, we believe that the high bone mass phenotype associated with the loss of sclerostin may be attributed, at least in part, to an increase in canonical Wnt signaling resulting from the reduction in sclerostin-mediated Wnt antagonism.     

Mutational Analysis of Sclerostin Shows Importance of the Flexible Loop and the Cystine-Knot for Wnt-Signaling Inhibition

The cystine-knot containing protein Sclerostin is an important negative regulator of bone growth and therefore represents a promising therapeutic target. It exerts its biological task by inhibiting the Wnt (wingless and int1) signaling pathway, which participates in bone formation by promoting the differentiation of mesenchymal stem cells to osteoblasts. The core structure of Sclerostin consists of three loops with the first and third loop (Finger 1 and Finger 2) forming a structured β-sheet and the second loop being unstructured and highly flexible. Biochemical data showed that the flexible loop is important for binding of Sclerostin to Wnt co-receptors of the low-density lipoprotein related-protein family (LRP), by interacting with the Wnt co-receptors LRP5 or -6 it inhibits Wnt signaling. To further examine the structural requirements for Wnt inhibition, we performed an extensive mutational study within all three loops of the Sclerostin core domain involving single and multiple mutations as well as truncation of important regions. By this approach we could confirm the importance of the second loop and especially of amino acids Asn92 and Ile94 for binding to LRP6. Based on a Sclerostin variant found in a Turkish family suffering from Sclerosteosis we generated a Sclerostin mutant with cysteines 84 and 142 exchanged thereby removing the third disulfide bond of the cystine-knot. This mutant binds to LRP6 with reduced binding affinity and also exhibits a strongly reduced inhibitory activity against Wnt1 thereby showing that also elements outside the flexible loop are important for inhibition of Wnt by Sclerostin. Additionally, we examined the effect of the mutations on the inhibition of two different Wnt proteins, Wnt3a and Wnt1. We could detect clear differences in the inhibition of these proteins, suggesting that the mechanism by which Sclerostin antagonizes Wnt1 and Wnt3a is fundamentally different.     

In silico discovery of quinoxaline derivatives as novel LRP5/6-sclerostin interaction inhibitors

The Wnt/β-catenin signaling pathway is a key regulator of bone homeostasis. Sclerostin act as an extracellular inhibitor of canonical Wnt signaling through high-affinity binding to the Wnt co-receptor LRP5/6. Disruption of the interaction between LRP5/6 and sclerostin has been recognized as a therapeutic target for osteoporosis. We identified a quinoxaline moiety as a new small-molecule inhibitor of the LRP5/6-sclerostin interaction through pharmacophore-based virtual screening, docking simulations, and in vitro assays. Structure-activity relationship studies and binding mode hypotheses were used to optimize the scaffold and yield the compound BMD4503-2, which recovered the downregulated activity of the Wnt/β-catenin signaling pathway by competitive binding to the LRP5/6-sclerostin complex. Overall, this study showed that the optimized structure-based drug design was a promising approach for the development of small-molecule inhibitors of the LRP5/6-sclerostin interaction. A novel scaffold offered considerable insights into the structural basis for binding to LRP5/6 and disruption of the sclerostin-mediated inhibition of Wnt signaling.     

Sequence and structural difference favors a distinct preference of Wnt3a binding with co-receptor LRP6

Wnt signaling pathway plays a key role in a wide array of development and physiological processes. Wnt proteins interact with two different co-receptors LRP5/6 and ROR 2, leading to different signal transductions in the cell. Though the Wnt family of proteins has high sequence similarity the specificity for particular co-receptor is not well understood. The choice of pathway is attributed to the binding of Wnt complex to the co-receptor. Our current study is a novel approach using homology modeling, docking, and structural alignment to unravel the structural differences between Wnt3a and Wnt5b binding to LRP6. The conservation of a protruding loop has been identified in Wnt3a protein indicating an enhanced ability of Wnt3a to bind to LRP5/6 against its counter parts. The docking studies have further substantiated the findings. This could potentially help us design and develop novel inhibitors targeting Wnt3a-LRP6 complex in specific tissues or disease states.     

Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling

Wnt/β-catenin signaling plays a central role in development and is also involved in a diverse array of diseases. Binding of Wnts to the coreceptors Frizzled and LRP6/5 leads to phosphorylation of PPPSPxS motifs in the LRP6/5 intracellular region and the inhibition of GSK3β bound to the scaffold protein Axin. However, it remains unknown how GSK3β is specifically inhibited upon Wnt stimulation. Here, we show that overexpression of the intracellular region of LRP6 containing a Ser/Thr rich cluster and a PPPSPxS motif impairs the activity of GSK3β in cells. Synthetic peptides containing the PPPSPxS motif strongly inhibit GSK3β in vitro only when they are phosphorylated. Microinjection of these peptides into Xenopus embryos confirms that the phosphorylated PPPSPxS motif potentiates Wnt-induced second body axis formation. In addition, we show that the Ser/Thr rich cluster of LRP6 plays an important role in LRP6 binding to GSK3β. These observations demonstrate that phosphorylated LRP6/5 both recruits and directly inhibits GSK3β using two distinct portions of its cytoplasmic sequence, and suggest a novel mechanism of activation in this signaling pathway.     

Wnt signal amplification via activity, cooperativity, and regulation of multiple intracellular PPPSP motifs in the Wnt co-receptor LRP6

Low density lipoprotein receptor-related protein 6 (LRP6) and its homologue LRP5 serve as Wnt co-receptors that are essential for the Wnt/beta-catenin pathway. Wnt activation of LRP6 leads to recruitment of the scaffolding protein Axin and inhibition of Axin-mediated phosphorylation/destruction of beta-catenin. We showed that five conserved PPPSP motifs in the LRP6 intracellular domain are required for LRP6 function, and mutation of these motifs together abolishes LRP6 signaling activity. We further showed that Wnt induces the phosphorylation of a prototypic PPPSP motif, which provides a docking site for Axin and is sufficient to transfer signaling activity to a heterologous receptor. However, the activity, regulation, and functionality of multiple PPPSP motifs in LRP6 have not been characterized. Here we provide a comprehensive analysis of all five PPPSP motifs in LRP6. We define the core amino acid residues of a prototypic PPPSP motif via alanine scanning mutagenesis and demonstrate that each of the five PPPSP motifs exhibits signaling and Axin binding activity in isolation. We generated two novel phosphorylation-specific antibodies to additional PPPSP motifs and show that Wnt induces phosphorylation of these motifs in the endogenous LRP6 through glycogen synthase kinase 3. Finally, we uncover the critical cooperativity of PPPSP motifs in the full-length LRP6 by demonstrating that LRP6 mutants lacking a single PPPSP motif display compromised function, whereas LRP6 mutants lacking two of the five PPPSP motifs are mostly inactive. This cooperativity appears to reflect the ability of PPPSP motifs to promote the phosphorylation of one another and to interact with Axin synergistically. These results establish the critical role and a common phosphorylation/activation mechanism for the PPPSP motifs in LRP6 and suggest that the conserved multiplicity and cooperativity of the PPPSP motifs represents a built-in amplifier for Wnt signaling by the LRP6 family of receptors.     

Negative regulation of LRP6 function by casein kinase I epsilon phosphorylation

Wnt signaling acts in part through the low density lipoprotein receptor-related transmembrane proteins LRP5 and LRP6 to regulate embryonic development and stem cell proliferation. Up-regulated signaling is associated with many forms of cancer. Casein kinase I epsilon (CKIepsilon) is a known component of the Wnt-beta-catenin signaling pathway. We find that CKIepsilon binds to LRP5 and LRP6 in vitro and in vivo and identify three CKIepsilon-specific phosphorylation sites in LRP6. Two of the identified phosphorylation sites, Ser1420 and Ser1430, influence Wnt signaling in vivo, since LRP6 with mutation of these sites is a more potent activator of both beta-catenin accumulation and Lef-1 reporter activity. Whereas Wnt3a regulates CKIepsilon kinase activity, LRP6 does not, placing CKIepsilon upstream of LRP6. Mutation of LRP6 Ser1420 and Ser1430 to alanine strengthens its interaction with axin, suggesting a mechanism by which CKIepsilon may negatively regulate Wnt signaling. The role of CKIepsilon is therefore more complex than was previously appreciated. Generation of active CKIepsilon may induce a negative feedback loop by phosphorylation of sites on LRP5/6 that modulate axin binding and hence beta-catenin degradation.     

Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.     

The C-Terminal Region Mesd Peptide Mimics Full-Length Mesd and Acts as an Inhibitor of Wnt/β-Catenin Signaling in Cancer Cells

While Mesd was discovered as a specialized molecular endoplasmic reticulum chaperone for the Wnt co-receptors LRP5 and LRP6, recombinant Mesd protein is able to bind to mature LRP5 and LRP6 on the cell surface and acts as a universal antagonist of LRP5/6 modulators. In our previous study, we found that the C-terminal region of Mesd, which is absent in sequences from invertebrates, is necessary and sufficient for binding to mature LRP6 on the cell surface. In the present studies, we further characterized the interaction between the C-terminal region Mesd peptide and LRP5/6. We found that Mesd C-terminal region-derived peptides block Mesd binding to LRP5 at the cell surface too. We also showed that there are two LRP5/6 binding sites within Mesd C-terminal region which contain several positively charged residues. Moreover, we demonstrated that the Mesd C-terminal region peptide, like the full-length Mesd protein, blocked Wnt 3A- and Rspodin1-induced Wnt/β-catenin signaling in LRP5- and LRP6- expressing cells, suppressed Wnt/β-catenin signaling in human breast HS578T cells and prostate cancer PC-3 cells, and inhibited cancer cell proliferation, although the full-length Mesd protein is more potent than its peptide. Finally, we found that treatment of the full-length Mesd protein and its C-terminal region peptide significantly increased chemotherapy agent Adriamycin-induced cytotoxicity in HS578T and PC-3 cells. Together, our results suggest that Mesd C-terminal region constitutes the major LRP5/6-binding domain, and that Mesd protein and its C-terminal region peptide have a potential therapeutic value in cancer.     

Discovery of potential sclerostin inhibitors from plants with loop2 region of sclerostin inhibition by interacting with residues outside Pro-Asn-Ala-Ile-Gly motif

Abstract

Sclerostin, an antagonist of the Wnt/β-catenin signaling pathway, was discovered as a potential therapeutic target for stimulating bone formation in osteoporosis. In this study, molecular docking was employed to predict the binding of 29 herbal compounds, which were reported as bone formation stimulators, to the loop2 region of sclerostin. Then, the 50 ns molecular dynamics (MD) simulation of the complexes between sclerostin and the top 10 hits obtained from molecular docking were carried out. Root mean square deviations (RMSDs) analysis of MD trajectories pointed out that all ligands-complexes remain stable throughout the duration of MD simulations. In addition, the molecular mechanics/generalized born surface area (MM/GBSA) binding free energy and energy decomposition analyses were determined. The results here suggested that baicalin is the most promising inhibitor of sclerostin. Interestingly, baicalin binds to sclerostin via the hydrophobic interaction with the amino acid residues on loop2 region but outside the Pro-Asn-Ala-Ile-Gly (PNAIG) motif, particularly the Arg-Gly-Lys-Trp-Trp-Arg (RGKWWR) motif. This finding could be a novel strategy for developing new sclerostin inhibitors in the future.

Communicated by Ramaswamy H. Sarma     

Dissecting molecular differences between Wnt coreceptors LRP5 and LRP6

Low-density lipoprotein receptor-related proteins 5 and 6 (LRP5 and LRP6) serve as Wnt co-receptors for the canonical β-catenin pathway. While LRP6 is essential for embryogenesis, both LRP5 and LRP6 play critical roles for skeletal remodeling, osteoporosis pathogenesis and cancer formation, making LRP5 and LRP6 key therapeutic targets for cancer and disease treatment. LRP5 and LRP6 each contain in the cytoplasmic domain five conserved PPPSPxS motifs that are pivotal for signaling and serve collectively as phosphorylation-dependent docking sites for the scaffolding protein Axin. However existing data suggest that LRP6 is more effective than LRP5 in transducing the Wnt signal. To understand the molecular basis that accounts for the different signaling activity of LRP5 and LRP6, we generated a series of chimeric receptors via swapping LRP5 and LRP6 cytoplasmic domains, LRP5C and LRP6C, and studied their Wnt signaling activity using biochemical and functional assays. We demonstrate that LRP6C exhibits strong signaling activity while LRP5C is much less active in cells. Recombinant LRP5C and LRP6C upon in vitro phosphorylation exhibit similar Axin-binding capability, suggesting that LRP5 and LRP6 differ in vivo at a step prior to Axin-binding, likely at receiving phosphorylation. We identified between the two most carboxyl PPPSPxS motifs an intervening "gap4" region that appears to account for much of the difference between LRP5C and LRP6C, and showed that alterations in this region are sufficient to enhance LRP5 PPPSPxS phosphorylation and signaling to levels comparable to LRP6 in cells. In addition we provide evidence that binding of phosphorylated LRP5 or LRP6 to Axin is likely direct and does not require the GSK3 kinase as a bridging intermediate as has been proposed. Our studies therefore uncover a new and important molecular tuning mechanism for differential regulation of LRP5 and LRP6 phosphorylation and signaling activity.     

LRP6 dimerization through its LDLR domain is required for robust canonical Wnt pathway activation

Canonical Wnt/β-catenin signaling pathway plays important roles in multiple aspects of cellular responses in development and diseases. It is currently thought that Wnt receptor Frizzled (Frz) exists separately to Wnt coreceptors LRP5 and LRP6 (LRP5/6), and that Wnt–Frz–LRP5/6 triple complex formation bridged by Wnt ligand is needed for canonical pathway activation. We recently showed that Frz and LRP5/6 interact with each other in the absence of Wnt ligand binding and this interaction maintains the Frz–LRP5/6 complex in an inactive state. Here, we further show that Wnt ligand stimulation induces conformational change of the Frz–LRP6 complex and leads to hexamer formation containing the core LDLR domain-mediated LRP6 homodimer that is stabilized by two pairs of Wnt3a and Frz8, that is, Wnt3a–Frz8–LRP6–LRP6–Frz8–Wnt3a. This LDLR-mediated LRP6 dimerization is essential for robust canonical Wnt pathway activation. Our study thus suggests a previously unrecognized mode of receptor interaction in Wnt signal initiation.