Mikhail V. Semenov

LDL receptor-related proteins 5 and 6 in Wnt/β-catenin signaling: Arrows point the way

Wnt signaling through the canonical β-catenin pathway plays essential roles in development and disease. Low-density-lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) in vertebrates, and their Drosophila ortholog Arrow, are single-span transmembrane proteins that are indispensable for Wnt/β-catenin signaling, and are likely to act as Wnt co-receptors. This review highlights recent progress and unresolved issues in understanding the function and regulation of Arrow/Lrp5/Lrp6 in Wnt signaling. We discuss Arrow/Lrp5/Lrp6 interactions with Wnt and the Frizzled family of Wnt receptors, and with the intracellular β-catenin degradation apparatus. We also discuss the regulation of Lrp5/Lrp6 by other extracellular ligands, and LRP5 mutations associated with familial osteoporosis and other disorders.

HEAD INDUCER DICKKOPF-1 IS A LIGAND FOR WNT CORECEPTOR LRP6

BACKGROUND Dickkopf-1 (Dkk-1) is a head inducer secreted from the vertebrate head organizer and induces anterior development by antagonizing Wnt signaling. Although several families of secreted antagonists have been shown to inhibit Wnt signal transduction by binding to Wnt, the molecular mechanism of Dkk-1 action is unknown. The Wnt family of secreted growth factors initiates signaling via the Frizzled (Fz) receptor and its candidate coreceptor, LDL receptor-related protein 6 (LRP6), presumably through Fz-LRP6 complex formation induced by Wnt. The significance of the Fz-LRP6 complex in signal transduction remains to be established. RESULTS We report that Dkk-1 is a high-affinity ligand for LRP6 and inhibits Wnt signaling by preventing Fz-LRP6 complex formation induced by Wnt. Dkk-1 binds neither Wnt nor Fz, nor does it affect Wnt-Fz interaction. Dkk-1 function in head induction and Wnt signaling inhibition strictly correlates with its ability to bind LRP6 and to disrupt the Fz-LRP6 association. LRP6 function and Dkk-1 inhibition appear to be specific for the Wnt/Fz beta-catenin pathway. CONCLUSIONS Our results demonstrate that Dkk-1 is an LRP6 ligand and inhibits Wnt signaling by blocking Wnt-induced Fz-LRP6 complex formation. Our findings thus reveal a novel mechanism for Wnt signal modulation. LRP6 is a Wnt coreceptor that appears to specify Wnt/Fz signaling to the beta-catenin pathway, and Dkk-1, distinct from Wnt binding antagonists, may be a specific inhibitor for Wnt/beta-catenin signaling. Our findings suggest that Wnt-Fz-LRP6 complex formation, but not Wnt-Fz interaction, triggers Wnt/beta-catenin signaling.

SnapShot: Noncanonical Wnt Signaling Pathways

Noncanonical Wnt signaling is defined here as Wnt- or Fz-initiated signaling that is independent of β-catenin transcriptional function. Noncanonical Wnt pathways are diverse and in many cases less characterized/defined. They are grouped into several categories for clarity and simplicity. Such classifications are not rigid as these pathways likely overlap with or intersect one another and are evolving.(A) Wnt/PCP (planar cell polarity) signaling. Vertebrate Wnt5a and Wnt11 initiate signaling via Fz, which may activate trimeric G proteins and Dvl (likely via phosphory-lation), and the assembly of Dvl/effector complexes. Multiple pathways downstream of Dvl regulate actin cytoskeleton/cell adhesion. The Dvl-Daam1-RhoA complex activates RhoA, which leads to Rock kinase activation and MRLC phosphorylation; Dvl-Rac1 complex activates JNK kinase, which may phosphorylate CapZIP/Dub; Dvl and XRNF185 (an E3 ligase) downregulate PXN protein level. Dvl recruitment to the membrane by Fz is regulated by kinases including Par1, CK1e, and PKCδ. Dvl-binding proteins/complexes—including Div, Inv (related to

LRP5 Mutations Linked to High Bone Mass Diseases Cause Reduced LRP5 Binding and Inhibition by SOST

The low density lipoprotein (LDL) receptor-related protein 5 (LRP5) is a co-receptor for Wnt proteins and a major regulator in bone homeostasis. Human genetic studies have shown that recessive loss-of-function mutations in LRP5 are linked to osteoporosis, while on the contrary, dominant missense LRP5 mutations are associated with high bone mass (HBM) diseases. All LRP5 HBM mutations are clustered in a single region in the LRP5 extracellular domain and presumably result in elevated Wnt signaling in bone forming cells. Here we show that LRP5 HBM mutant proteins exhibit reduced binding to a secreted bone-specific LRP5 antagonist, SOST, and consequently are more refractory to inhibition by SOST. As loss-of-function mutations in the SOST gene are associated with Sclerosteosis, another disorder of excessive bone growth, our study suggests that the SOST-LRP5 antagonistic interaction plays a central role in bone mass regulation and may represent a nodal point for therapeutic intervention for osteoporosis and other bone diseases.

R-spondin1 Is a High Affinity Ligand for LRP6 and Induces LRP6 Phosphorylation and β-Catenin Signaling

R-spondin proteins are newly identified secreted molecules that activate β-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 (Kd = 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.

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.

Identification of signal peptide domain SOST mutations in autosomal dominant craniodiaphyseal dysplasia.

Sclerosteosis and Van Buchem disease are related recessive sclerosing bone dysplasias caused by alterations in the SOST gene. We tested the hypothesis that craniodiaphyseal dysplasia (CDD) (MIM 122860), an extremely rare sclerosing bone dysplasia resulting facial distortion referred to as “leontiasis ossea”, could also be caused by SOST mutations. We discovered mutations c.61G>A (Val21Met) and c.61G>T (Val21Leu) two children with CDD. As these mutations are located in the secretion signal of the SOST gene, we tested their effect on secretion by transfecting the mutant constructs into 293E cells. Intriguingly, these mutations greatly reduced the secretion of SOST. We conclude that CDD, the most severe form of sclerotic bone disease, is part of a spectrum of disease caused by mutations in SOST. Unlike the other SOST-related conditions, sclerosteosis and Van Buchem disease that are inherited as recessive traits seem to be caused by a dominant negative mechanism.

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.

Wnt-1 expression in PC12 cells induces exon 15 deletion and expression of L-APP.

The metabolism of amyloid precursor protein (APP) is central to Alzheimers disease pathogenesis. Recent data have linked APP and presenilin to the Wnt/wingless signaling pathway. To assess affects of Wnt stimulation on APP isoform expression, we infected PC12 cells and C57MG cells with a retrovirus containing murine Wnt-1. In PC12 cells, Wnt-1 expression is associated with induction of exon 15 deletion from APP mRNA and expression of L-APP. Our data suggest that APP isoform expression is regulated, in part, by the Wnt/wingless signaling pathway.