DKK1 antagonizes Wnt signaling without promotion of LRP6 internalization and degradation

DKK1 is a secreted protein that antagonizes Wnt signaling and plays essential roles in vertebrate embryogenesis including head induction, skeletal development, and limb patterning. DKK1 is also implicated in osteoporosis, arthritis, and cancer and represents a potential therapeutic target for the treatment of these diseases. DKK1 is a high affinity antagonistic ligand for LRP6, which is a Wnt coreceptor that acts together with the Frizzled serpentine receptor to initiate Wnt signal transduction. Two different models have been proposed to account for the mechanism by which DKK1 antagonizes LRP6 function. One model suggests that DKK1 binding to LRP6 disrupts Wnt-induced Frizzled-LRP6 complex formation, whereas the other model proposes that DKK1 interaction with LRP6 promotes LRP6 internalization and degradation, thereby reducing the cell surface LRP6 level. To clarify the molecular basis of DKK1 action, we examined how DKK1 affects the endogenous LRP6 in several mammalian cell lines including mouse embryonic fibroblasts. Here we show that DKK1 inhibits Wnt signaling but induces neither LRP6 down-regulation from the cell surface nor reduction of total LRP6 protein level and that DKK1 has no effect on the rate of continuous internalization of LRP6 and the half-life (about 4.7 h) of LRP6. We conclude that DKK1 inhibition of LRP6 is independent of LRP6 internalization and degradation.     

The First Propeller Domain of LRP6 Regulates Sensitivity to DKK1

The Wnt coreceptor LRP6 is required for canonical Wnt signaling. To understand the molecular regulation of LRP6 function, we generated a series of monoclonal antibodies against the extra cellular domain (ECD) of LRP6 and selected a high-affinity mAb (mAb135) that recognizes cell surface expression of endogenous LRP6. mAb135 enhanced Wnt dependent TCF reporter activation and antagonized DKK1 dependent inhibition of Wnt3A signaling, suggesting a role in modulation of LRP6 function. Detailed analysis of LRP6 domain mutants identified Ser 243 in the first propeller domain of LRP6 as a critical residue for mAb135 binding, implicating this domain in regulating the sensitivity of LRP6 to DKK1. In agreement with this notion, mAb135 directly disrupted the interaction of DKK1 with recombinant ECD LRP6 and a truncated form of the LRP6 ECD containing only repeats 1 and 2. Finally, we found that mAb135 completely protected LRP6 from DKK1 dependent internalization. Together, these results identify the first propeller domain as a novel regulatory domain for DKK1 binding to LRP6 and show that mAb against the first propeller domain of LRP6 can be used to modulate this interaction.     

The LDL receptor-related protein LRP6 mediates internalization and lethality of anthrax toxin

Toxins produced by Bacillus anthracis and other microbial pathogens require functions of host cell genes to yield toxic effects. Here we show that low density lipoprotein receptor-related protein 6 (LRP6), previously known to be a coreceptor for the Wnt signaling pathway, is required for anthrax toxin lethality in mammalian cells. Downregulation of LRP6 or coexpression of a truncated LRP6 dominant-negative peptide inhibited cellular uptake of complexes containing the protective antigen (PA) carrier of anthrax toxin moieties and protected targeted cells from death, as did antibodies against epitopes in the LRP6 extracellular domain. Fluorescence microscopy and biochemical analyses showed that LRP6 enables toxin internalization by interacting at the cell surface with PA receptors TEM8/ATR and/or CMG2 to form a multicomponent complex that enters cells upon PA binding. Our results, which reveal a previously unsuspected biological role for LRP6, identify LRP6 as a potential target for countermeasures against anthrax toxin lethality.     

Functional interactions between anthrax toxin receptors and the WNT signalling protein LRP6

To exert its activity, anthrax toxin must be endocytosed and its enzymatic toxic subunits delivered to the cytoplasm. It has been proposed that, in addition to the anthrax toxin receptors (ATRs), lipoprotein-receptor-related protein 6 (LRP6), known for its role in Wnt signalling, is also required for toxin endocytosis. These findings have however been challenged. We show that LRP6 can indeed form a complex with ATRs, and that this interaction plays a role both in Wnt signalling and in anthrax toxin endocytosis. We found that ATRs control the levels of LRP6 in cells, and thus the Wnt signalling capacity. RNAi against ATRs indeed led to a drastic decrease in LRP6 levels and a subsequent drop in Wnt signalling. Conversely, LRP6 plays a role in anthrax toxin endocytosis, but is not essential. We indeed found that toxin binding triggered tyrosine phosphorylation of LRP6, induced its redistribution into detergent-resistant domains, and its subsequent endocytosis. RNAis against LRP6 strongly delayed toxin endocytosis. As the physiological role of ATRs is probably to interact with the extracellular matrix, our findings raise the interesting possibility that, through the ATR-LRP6 interaction, adhesion to the extracellular matrix could locally control Wnt signalling.     

In silico studying of the whole protein structure and dynamics of Dickkopf family members showed that N-terminal domain of Dickkopf 2 in contrary to other Dickkopfs facilitates its interaction with low density lipoprotein receptor related protein 5/6

Wnt (Wingless Int) signaling pathway has been known to be dysregulated in several human cancers, especially colorectal cancer (CRC). The Dickkopf (DKK) family which consists of four secreted proteins in vertebrates (DKK 1, 2, 3, 4) is one of the most critical antagonist families for Wnt signaling pathway. They typically antagonize Wnt/β-catenin signaling by binding and inhibiting Wnt co-receptors, LRP5/6 (low density lipoprotein receptor related protein 5/6). However, except for DKK1 (Dickkopf 1), details about structure and function of the members of this family are poorly defined. In this study, main Dickkopf family members were analyzed structurally, using protein structure prediction tools, molecular dynamics (MD), molecular docking and energy analyses. Three dimensional structure of whole DKKs was predicted and their interaction with LRP6 was investigated in detail. The results indicated that in DKK family members, a considerable diversity, in the case of structure, activity and physicochemical properties was seen. This diversity was more profound in DKK3 (Dickkopf3). Interestingly, the interaction mode of DKK2 (Dickkopf2) with its receptor, LRP6, was shown to be substantially different from other Dickkopf family members while N-terminal region of this ligand was also involved in the binding to the LRP6-P3P4. Moreover, the cysteine-rich domain 2 (CRD2) of DKK1 and DKK3 had a higher binding affinity to LRP6 in comparison with the whole protein structures.

Communicated by Ramaswamy H. Sarma     

LRP5 and LRP6 are not required for protective antigen-mediated internalization or lethality of anthrax lethal toxin

Anthrax toxin (AnTx) plays a key role in the pathogenesis of anthrax. AnTx is composed of three proteins: protective antigen (PA), edema factor, and lethal factor (LF). PA is not toxic but serves to bind cells and translocate the toxic edema factor or LF moieties to the cytosol. Recently, the low-density lipoprotein receptor-related protein LRP6 has been reported to mediate internalization and lethality of AnTx. Based on its similarity to LRP6, we hypothesized that LRP5 may also play a role in cellular uptake of AnTx. We assayed PA-dependent uptake of anthrax LF or a cytotoxic LF fusion protein (FP59) in cells and mice harboring targeted deletions of Lrp5 or Lrp6. Unexpectedly, we observed that uptake was unaltered in the presence or absence of either Lrp5 or Lrp6 expression. Moreover, we observed efficient PA-mediated uptake into anthrax toxin receptor (ANTXR)-deficient Chinese hamster ovary cells (PR230) that had been stably engineered to express either human ANTXR1 or human ANTXR2 in the presence or absence of siRNA specific for LRP5 or LRP6. Our results demonstrate that neither LRP5 nor LRP6 is necessary for PA-mediated internalization or lethality of anthrax lethal toxin.     

R-Spondin family members regulate the Wnt pathway by a common mechanism

The R-Spondin (RSpo) family of secreted proteins is implicated in the activation of the Wnt signaling pathway. Despite the high structural homology between the four members, expression patterns and phenotypes in knockout mice have demonstrated striking differences. Here we dissected and compared the molecular and cellular function of all RSpo family members. Although all four RSpo proteins activate the canonical Wnt pathway, RSpo2 and 3 are more potent than RSpo1, whereas RSpo4 is relatively inactive. All RSpo members require Wnt ligands and LRP6 for activity and amplify signaling of Wnt3A, Wnt1, and Wnt7A, suggesting that RSpo proteins are general regulators of canonical Wnt signaling. Like RSpo1, RSpo2-4 antagonize DKK1 activity by interfering with DKK1 mediated LRP6 and Kremen association. Analysis of RSpo deletion mutants indicates that the cysteine-rich furin domains are sufficient and essential for the amplification of Wnt signaling and inhibition of DKK1, suggesting that Wnt amplification by RSpo proteins may be a direct consequence of DKK1 inhibition. Together, these findings indicate that RSpo proteins modulate the Wnt pathway by a common mechanism and suggest that coexpression with specific Wnt ligands and DKK1 may determine their biological specificity in vivo.     

The LRP5 high-bone-mass G171V mutation disrupts LRP5 interaction with Mesd

The mechanism by which the high-bone-mass (HBM) mutation (G171V) of the Wnt coreceptor LRP5 regulates canonical Wnt signaling was investigated. The mutation was previously shown to reduce DKK1-mediated antagonism, suggesting that the first YWTD repeat domain where G171 is located may be responsible for DKK-mediated antagonism. However, we found that the third YWTD repeat, but not the first repeat domain, is required for DKK1-mediated antagonism. Instead, we found that the G171V mutation disrupted the interaction of LRP5 with Mesd, a chaperone protein for LRP5/6 that is required for transport of the coreceptors to cell surfaces, resulting in fewer LRP5 molecules on the cell surface. Although the reduction in the number of cell surface LRP5 molecules led to a reduction in Wnt signaling in a paracrine paradigm, the mutation did not appear to affect the activity of coexpressed Wnt in an autocrine paradigm. Together with the observation that osteoblast cells produce autocrine canonical Wnt, Wnt7b, and that osteocytes produce paracrine DKK1, we think that the G171V mutation may cause an increase in Wnt activity in osteoblasts by reducing the number of targets for paracrine DKK1 to antagonize without affecting the activity of autocrine Wnt.     

Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of beta-catenin signaling

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, we found that Wnt3a acts in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively.     

Dkk1 Stabilizes Wnt Co-Receptor LRP6: Implication for Wnt Ligand-Induced LRP6 Down-Regulation

Background

The low density lipoprotein receptor-related protein-6 (LRP6) is an essential co-receptor for canonical Wnt signaling. Dickkopf 1 (Dkk1), a major secreted Wnt signaling antagonist, binds to LRP6 with high affinity and prevents the Frizzled-Wnt-LRP6 complex formation in response to Wnts. Previous studies have demonstrated that Dkk1 promotes LRP6 internalization and degradation when it forms a ternary complex with the cell surface receptor Kremen.

Methodology/Principal Findings

In the present study, we found that transfected Dkk1 induces LRP6 accumulation while inhibiting Wnt/LRP6 signaling. Treatment with Dkk1-conditioned medium or recombinant Dkk1 protein stabilized LRP6 with a prolonged half-life and induces LRP6 accumulation both at the cell surface and in endosomes. We also demonstrated that Kremen2 co-expression abrogated the effect of Dkk1 on LRP6 accumulation, indicating that the effect of Kremen2 is dominant over Dkk1 regulation of LRP6. Furthermore, we found that Wnt3A treatment induces LRP6 down-regulation, an effect paralleled with a Wnt/LRP6 signaling decay, and that Dkk1 treatment blocked Wnt3A-induced LRP6 down-regulation. Finally, we found that LRP6 turnover was blocked by an inhibitor of caveolae-mediated endocytosis.

Conclusions/Significance

Our results reveal a novel role for Dkk1 in preventing Wnt ligand-induced LRP6 down-regulation and contribute significantly to our understanding of Dkk1 function in Wnt/LRP6 signaling.     

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.     

Clathrin and AP2 are required for PtdIns(4,5)P2-mediated formation of LRP6 signalosomes

Canonical Wnt signaling is initiated by the binding of Wnt proteins to their receptors, low-density lipoprotein-related protein 5 and 6 (LRP5/6) and frizzled proteins, leading to phosphatidylinositol (4,5)bisphosphate (PtdIns(4,5)P₂) production, signalosome formation, and LRP phosphorylation. However, the mechanism by which PtdIns(4,5)P₂ regulates the signalosome formation remains unclear. Here we show that clathrin and adaptor protein 2 (AP2) were part of the LRP6 signalosomes. The presence of clathrin and AP2 in the LRP6 signalosomes depended on PtdIns(4,5)P₂, and both clathrin and AP2 were required for the formation of LRP6 signalosomes. In addition, WNT3A-induced LRP6 signalosomes were primarily localized at cell surfaces, and WNT3A did not induce marked LRP6 internalization. However, rapid PtdIns(4,5)P₂ hydrolysis induced artificially after WNT3A stimulation could lead to marked LRP6 internalization. Moreover, we observed WNT3A-induced LRP6 and clathrin clustering at cell surfaces using super-resolution fluorescence microscopy. Therefore, we conclude that PtdIns(4,5)P₂ promotes the assembly of LRP6 signalosomes via the recruitment of AP2 and clathrin and that LRP6 internalization may not be a prerequisite for Wnt signaling to β-catenin stabilization.     

G Protein-coupled Receptor Kinases Phosphorylate LRP6 in the Wnt Pathway

Wnt ligands conduct their functions in canonical Wnt signaling by binding to two receptors, the single transmembrane low density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) and seven transmembrane (7TM) Frizzled receptors. Subsequently, phosphorylation of serine/threonine residues within five repeating signature PPPSP motifs on LRP6 is responsible for LRP6 activation. GSK3β, a cytosolic kinase for phosphorylation of a downstream effector β-catenin, was proposed to participate in such LRP6 phosphorylation. Here, we report a new class of membrane-associated kinases for LRP6 phosphorylation. We found that G protein-coupled receptor kinases 5 and 6 (GRK5/6), traditionally known to phosphorylate and desensitize 7TM G protein-coupled receptors, directly phosphorylate the PPPSP motifs on single transmembrane LRP6 and regulate Wnt/LRP6 signaling. GRK5/6-induced LRP6 activation is inhibited by the LRP6 antagonist Dickkopf. Depletion of GRK5 markedly reduces Wnt3A-stimulated LRP6 phosphorylation in cells. In zebrafish, functional knock-down of GRK5 results in reduced Wnt signaling, analogous to LRP6 knock-down, as assessed by decreased abundance of β-catenin and lowered expression of the Wnt target genes cdx4, vent, and axin2. Expression of GRK5 rescues the diminished β-catenin and axin2 response caused by GRK5 depletion. Thus, our findings identify GRK5/6 as novel kinases for the single transmembrane receptor LRP6 during Wnt signaling.     

Computationally Design of Inhibitory Peptides Against Wnt Signaling Pathway: In Silico Insight on Complex of DKK1 and LRP6

Wnt signaling pathway plays a major role in the regulation of cell proliferation, migration, tissue homeostasis, tumor progression and cancer. This pathway can be antagonized by different proteins such as DKK proteins, which disrupt the initiatory complex (Frizzled–LRP6 complex). Therefore, interruption of its formation could be a promising strategy for the design of Low-density lipoprotein receptor-Related Protein 6 (LRP6) inhibitors. A computational study was conducted in order to assist in the design of inhibitory peptides against LRP6 as co-receptor of frizzled. Twelve fragments as peptide derivatives of natural ligand of LRP6 receptor (DKK1) were designed using the information from the analysis of the DKK1_C/LRP6 complex, hot spot residues and the secondary structure. These fragments were based on cys2 domain of DKK1. The designed peptides were energy minimized by molecular dynamics simulations in the presence and absence of LRP6 receptor and their binding affinities were investigated via molecular docking using ClusPro, HADDOCK and PRODIGY webservers. Finally, the stability and free energy of binding in peptides were calculated by FoldX software. The results showed that four designed peptides had the highest affinity (the interaction energy: ?10.2867, ?10.1388, ?7.94339 and ?7.57536 kcal/mol) to interact with the receptor which showed the most interacting residues and the lowest free energy of binding. Also, the RMSD, RMSF and RoG of the protein–peptide complex exhibited less structural fluctuations which can be linked to the stability of peptides associated to the receptor. These peptides may be considered as candidates for inhibiting Wnt signaling pathway through LRP6 receptor.     

Evidence against a human cell-specific role for LRP6 in anthrax toxin entry

The role of the cellular protein LRP6 in anthrax toxin entry is controversial. Previous studies showed that LRP6 was important for efficient intoxication of human M2182 prostate carcinoma cells but other studies performed with cells from gene-knockout mice demonstrated no role for either LRP6 or the related LRP5 protein in anthrax toxin entry. One possible explanation for this discrepancy is that LRP6 may be important for anthrax toxin entry into human, but not mouse, cells. To test this idea we have investigated the effect of knocking down LRP6 or LRP5 expression with siRNAs in human HeLa cells. We show here that efficient knockdown of either LRP6, LRP5, or both proteins has no influence on the kinetics of anthrax lethal toxin entry or MEK1 substrate cleavage in these cells. These data argue against a human-specific role for LRP6 in anthrax toxin entry and suggest instead that involvement of this protein may be restricted to certain cell types independently of their species of origin.     

R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and beta-catenin signaling

R-spondin proteins are newly identified secreted molecules that activate beta-catenin signaling. However, the mechanism of R-spondin action and its relationship with Wnt signaling remain unclear. Here we show that human R-spondin1 (hRspo1) is a high affinity ligand for the Wnt co-receptor LRP6 (K(d) = 1.2 nm). hRspo1 induces glycogen synthase kinase 3-dependent phosphorylation and activation of LRP6. DKK1, an LRP6 antagonist, inhibits hRspo1-induced LRP6 phosphorylation. We further demonstrate that hRspo1 synergizes with Frizzled5 in Xenopus axis induction assays and induces the phosphorylation of Dishevelled, a cytoplasmic component downstream of Frizzled function. Our study reveals interesting similarity and distinction between Wnt and R-spondin signaling.     

The Low-density Lipoprotein Receptor-related Protein 6 Pathway in the Treatment of Intestinal Barrier Dysfunction Induced by Hypoxia and Intestinal Microbiota through the Wnt/β-catenin Pathway

Our study is to explore the key molecular of Low-density lipoprotein receptor-related protein 6 (LRP6) and the related Wnt/β-catenin pathway regulated by LRP6 during the intestinal barrier dysfunction. Colorectal protein profile analysis showed that LRP6 expression was decreased in dextran sulfate sodium (DSS)-induced colitis mice, and mice received fecal bacteria transplantation from stroke patients. Mice with intestinal hypoxia and intestinal epithelial cells cultured in hypoxia showed decreased expression of LRP6. Overexpression of LPR6 or its N-terminus rescued the Wnt/β-catenin signaling pathway which was inhibited by hypoxia and endoplasmic reticulum stress. In mice overexpressing of LRP6, the expression of β-catenin and DKK1 increased, Bcl2 decreased, and Bax increased. Mice with LRP6 knockout showed an opposite trend, and the expression of Claudin2, Occludin and ZO-1 decreased. Two drugs, curcumin and auranofin could alleviate intestinal barrier damage in DSS-induced colitis mice by targeting LRP-6. Therefore, gut microbiota dysbiosis and hypoxia can inhibit the LRP6 and Wnt/β-catenin pathway, and drugs targeting LRP6 can protect the intestinal barrier.