LRP5 and Wnt Signaling: A Union Made for Bone

The identification of mutations in the human low-density lipoprotein (LDL) receptor-related protein 5 (LRP5) gene that give rise to conditions of low bone mass1, 2 and increased bone mass3-5 has brought what just a few years ago were two seemingly unrelated fields crashing together, namely bone biology and Wnt signaling. It is now clear that the Wnt signaling pathway is another key pathway involved in the regulation of bone mass. This is not to diminish the importance of any other pathway that is known to play a role; rather, this discovery only highlights how little we probably really know about bone mass regulation. Certainly, a big challenge we now face will be to integrate the Wnt signaling pathway with all of its complexities and subtleties into the already complex nature of the other pathways that have been studied, with the ultimate goal of developing a composite understanding of their collective roles and interactions. In this article, we will summarize the main points of our current understanding of LRP5, Wnt's and Wnt signaling, and the role they may play in bone. This is by no means comprehensive, and there are more unanswered questions than answers. There are currently at least 13 known members of the LDL receptor family that serve a variety of cargo (transport) and cell signaling functions in various tissues and/or cell types.6 All members of the family are single-pass transmembrane proteins that contain highly conserved motifs; most notably, all contain copies of a complement-like cysteine-rich repeat motif (the LDL repeat). In addition, most contain a tyrosine-tryptophan-threonine-aspartate (YWTD) amino acid repeat that forms a β-propeller structure critical for proper function (ligand binding). Another common feature of most members is the presence of an epidermal growth factor (EGF)-like repeat motif and an NPxY sequence in the cytoplasmic tail that serves as a signal sequence for endocytosis of the receptor. The parental member of the family, the LDL receptor, has been well studied and characterized because of its central role in the pathophysiology of familial hypercholesterolemia.7 The elucidation of the pathway through which the LDL receptor regulates the binding, endocytosis, and degradation of cholesterol-rich lipoproteins is a classic discovery in the fields of cell and molecular biology and has been reviewed extensively.8 Blacklow and colleagues have extensively studied the structure of the LDL receptor in terms of the functional properties of the various motifs in the extracellular domain of the protein and how mutations observed in familial hypercholesterolemia alter receptor folding and function.9-12 His group has also reported the crystal structure of the YWTD-EGF domain pair.13 LRP5 was cloned independently by three different groups in 1998.14-16 In a screen for loci genetically linked to type I diabetes, evidence was obtained that the IDDM4 locus on chromosome 11q13 contained a major susceptibility gene.17, 18 Hey et al.16 subsequently identified a novel member of the LDL receptor family, which they termed LRP5, as a candidate gene for the IDDM4 locus (although it now seems that mutations in LRP5 do not contribute to the IDDM phenotype). They cloned both the human and mouse LRP5 gene. Later this group identified a closely homologous gene to LRP5 in human and mouse that they called LRP6.19 LRP6 is located on chromosome 12p11.2-p13.3.19 The genomic structure of LRP5 has been described.20 The genomic structure contains 23 exons and spans a region of 160 kb. Kim et al.14 and Dong et al.15 also independently cloned LRP5 in 1998. Kim et al.14 identified the human and rabbit LRP5 gene as a result of their screen for new receptors that might play a role in apo-E containing lipoproteins clearance. Dong et al.15 identified a gene they called LR3 from a screen of a human osteoblast cDNA library. Subsequent sequence analysis showed that their LR3 gene was the same as the LRP5 gene cloned by Hey et al.16 and Kim et al.14 LRP5 and 6 are homologous to the Drosophila protein, Arrow.21 LRP5 and 6 are unique with respect to other members of the LDL receptor family in that they do not contain the internalization signal sequence for endocytosis. The LRP5 protein contains 1615 amino acids, whereas LRP6 contains 1613 amino acids. Overall homology between LRP5 and 6 at the nucleotide level is 64%, whereas coding sequence homology is 71%.19 The extracellular portion consists of four EGF-like repeats, each separated by six YWTD spacer domains. These EGF-like repeats are implicated in protein-protein interactions because they contribute to ligand binding in some LRPs.22 In addition, there are three LDLR type A repeats between the EGF-like repeats and the membrane. LDLR repeats are required for binding of the LDL particle by the LDL receptor and so may also contribute to ligand-receptor interactions.22 The crystal structure of the EGF domain pair in the LDL receptor indicates that the YWTD repeats form a six-bladed β-propeller structure.13 A recent study of the nidogen β-propeller domain that binds laminin by Takagi et al.23 has revealed that the β-propeller domain of LRP5 closely resembles the nidogen binding site for LE4 and suggests a structural paradigm for high affinity ligand-binding to the YWTD β-propeller 1 of LRP5. As will be discussed later, all of the mutations in LRP5 that give rise to conditions of increased bone mass3-5 are found in this first YWTD β-propeller. Strickland et al.6 have recently published a model showing and comparing the domain structures for all members of the LDL receptor family, including LRP5/6 in the review. The Wnt proteins are secreted from cells, but in a lipid-modified and very hydrophobic form that can only be solubilized in detergent.24, 25 The lipid modification consists of a covalent attachment of a palmitate to a conserved cysteine near the amino terminus of the Wnt protein. Enzymatic removal of the palmitate results in loss of activity, and together with mutational analysis of the modified cysteine, indicates that the palmitoylation is important for Wnt signaling. There is genetic evidence for a dedicated enzyme, encoded by the Drosophila gene porcupine, catalyzing Wnt palmitoylation.26 It is not known whether secreted forms of Wnt's are bound to carrier molecules, but it is possible that such carriers exist, in particular, molecules that may bind to the lipid to generate a complex that is more water soluble and released from cells. In addition to lipidation, Wnt's are also modified by glycosylation, but this modification is not essential for activity.27 Given the general importance of the LDL receptor family for the uptake of lipids and other macromolecules, the relationship between LRPs and the response of cells to Wnt's is intriguing. Because Wnt's are lipid-modified and dependent on the lipid for signaling activity, it is tempting to speculate that LRPs specifically interact with the lipid on the Wnt and thereby promote signaling. While this is certainly an attractive possibility, there is yet no evidence supporting it, and it is equally likely that the interaction is indirect and involves an apoprotein that is bound to the lipid on the Wnt molecule. Further biochemical experiments are necessary to distinguish between these mechanisms. The extracellular portion of LRP5/6 bind several different molecules, principally the Wnt's and Dkk's, and the co-receptor protein, Frizzled (Fz; Fig. 1). The Fz transmembrane receptor28, 29 consists of an extracellular cysteine rich domain (CRD) that serves as a ligand-binding unit,30, 31 and a 7 transmembrane signaling portion. Using a deletion construct approach, the binding of Dkk-1 to LRP6 has been shown to occur through interaction with the third and fourth EGF-like repeat domains, and binding to Fz occurs with the EGF domain 1 and 2 of the receptor in a Wnt-dependent manner.32, 33 The multiple levels of extracellular regulation of LRP5/6/arrow and modulation of Wnt signaling. The binding of Wnt ligand to the frizzled and LRP5/6/arrow receptor leads to activation of the β-catenin signaling pathway (see Fig. 2). The binding of the Wnt antagonist Dickkopf (Dkk-1) results in the formation of a ternary complex with the single pass transmembrane protein, kremen, which results in LRP5/6/arrow internalization and degradation, thus removing LRP5/6/arrow from being able to interact with Wnt and frizzled. Secreted frizzled-related proteins (sFRPs) are also capable of binding Wnt and thus preventing it from binding to the LRP5/6/arrow-frizzled receptor. sFRP has also been shown to bind to RANKL and prevent its interaction with RANK, thereby preventing osteoclastogenesis. Wnt signaling is required for a diversity of developmental events including mesoderm induction, organogenesis, CNS organization and limb patterning.34-37 In addition, a number of Wnt's have been implicated in vertebrate skeletal development. For example, there is evidence that Wnt3a, Wnt4, Wnt5a, Wnt5b, and Wnt7a all have important roles in chondrogenesis.37-41 Another member of the Wnt family, Wnt9A (formerly Wnt14), can induce morphological and molecular signs of joint formation when inappropriately expressed, indicating that Wnt9A plays a crucial role in the initiation of synovial joint development.42 Wnt9A expression can also lead to the arrests and reversal of chondrogenic differentiation in vitro.42 Thus, Wnt's have various functions during the formation and the patterning of the skeleton. Recent evidence (O MacDougald, personal communication, 2004) has suggested an important role for Wnt10b in both bone and adipose tissue. Notably, activation of the canonical Wnt pathway by Wnt10b blocks adipogenesis in vitro and adipose tissue development in mice and increases osteoblastogenesis in vitro and trabecular bone mass in mice. Also, Wnt10b−/− mice have decreased trabecular mass. However, the natural ligand for LRP5/6 in bone remains unproven at this time and is a major unanswered question. Wnt's also bind to the secreted frizzled related proteins (sFRPs),43-45 which can modulate Wnt activity (Fig. 1). These proteins contain a cysteine rich domain (CRD) that is highly homologous to the CRD in the Fz receptors, but lack the transmembrane domain and are secreted as extracellular proteins. Hausler et al. have shown that sFRP-1 also binds to RANKL, which raises the possibility that analogous to the binding of RANKL by osteoprotegerin (OPG),46 sFRP-1 may regulate osteoclastogenesis. In general, members of the LDL receptor family have roles in the endocytic pathway and mediate the uptake of macromolecules. Examples are apolipoproteins endocytosed through clathrin-coated pits.22, 47 Mice carrying mutations in members of the LDL receptor family (the VLDL and ApoER2 receptors) have phenotypes similar to mouse mutant for reelin, indicating that LDL receptors can participate in specific signaling pathways.48 Initially, genetic evidence implicated the Drosophila arrow gene and its mammalian homolog LRP6 as having phenotypes very similar to Wnt phenotypes.21, 49 Genetic epistasis experiments in Drosophila have placed the requirement for Arrow/LRP5/6 downstream of the Wnt gene wingless and upstream of cytoplasmic signaling components, implying Arrow/LRP5/6 functions in parallel to or downstream of Fz.21 The intracellular region of the LRPs is highly enriched in proline, indicating that the cytoplasmic tail may serve as a docking site for SH3-domain containing proteins, although such proteins have not been identified. These proteins also contain putative internalization sequences within their intracellular domains. While genetic evidence has implicated LRP6 in Wnt signaling, analysis of different mammalian cell lines indicates that LRP5 is also involved in the canonical Wnt signal transduction pathway.1, 50 Other cell culture experiments using a Lef/TCF-dependent Luciferase assay show that Arrow/LRP5/6 functions cooperatively with Fz to stimulate the β-catenin pathway in response to different Wnt proteins.51 Biochemical analysis of human LRP6 shows that the extracellular portion binds to mouse Fz8 in a Wnt1-dependent manner.32 These data invite the idea that Wnt may initiate signal transduction by simply bringing LRP5/6 and Fz into close apposition. Furthermore, FRET analysis has shown a Wnt-dependent interaction between LRP5 and Axin.33 More recently, Tamai et al.52 have shown that a PPPSP motif, which is reiterated five times in the LRP5/6/Arrow intracellular domain, is necessary for Wnt signaling. In addition, Wnt signaling leads to phosphorylation of the PPPSP motif, which then generates an inducible binding site for Axin (Fig. 2). The observed Axin recruitment to LRP5/6 provides the first evidence of a distinct connection between extracellular receptor activation and intracellular signaling events. In view of the evidence that Fz can bind Dsh and that Dsh and Axin can directly contact each other through their DIX domains,53 it is tempting to postulate that Wnt signaling leads to a higher order complex at the membrane, containing the two receptors plus Axin and Dsh54-56 (Fig. 2). The binding of Wnt to the LRP5/6/arrow-frizzled receptor leads to recruitment of Axin to the cytoplasmic tail of LRP and possibly to a complex between Axin and Dishevelled (Dsh). Dishevelled inhibits glycogen synthase kinase-3β (GSK3β) from phosphorylating β-catenin. The combination of Axin binding to LRP5/6/arrow and inhibition of GSK3β causes the release of β-catenin from the Axin-APC-GSK3β complex, on which β-catenin is stabilized to enter the nucleus and interact with TCF. The current model of the Wnt canonical signaling pathway is that activation of cells by Wnt signaling initiates a cascade of intracellular events triggered by the cytoplasmic protein Dishevelled (Dsh) interacting directly with the Fz receptor54, 56 (Fig. 2). In the absence of a Wnt signal, β-catenin is localized to the cytoplasmic β-catenin destruction complex, which includes adenomatous polyposis coli (APC), glycogen synthase kinase (GSK)-3β, and Axin proteins. APC and Axin function as critical scaffold proteins that allow GSK3β to bind and phosphorylate β-catenin, thus tagging the protein for degradation through the ubiquitin/proteasome pathway.55, 57-59 On Wnt binding to the co-receptor complex, Dsh activation leads to the phosphorylation of GSK3β, thereby inhibiting the phosphorylation of β-catenin. The cytoplasmic tail of the Lrp5/6 binds to Axin, which facilitates dissociation of the destruction complex and leads to increased intracellular levels of β-catenin.50 This results in β-catenin stabilization and subsequent nuclear translocation (Fig. 2). Once β-catenin is localized to the nucleus, it interacts with Lef1/TCF transcription factors to initiate target gene activation.57 The specificity of LRP5/6/arrow in the Wnt pathway is further illustrated by the identification, both in mice and flies, of genes that are required as ER chaperones for transport and folding for LRPs. These chaperone genes, Boca and Mesd, have phenotypes very similar to Wnt pathway components.60, 61 In addition to its role in Wnt reception at the membrane, LRP5/6 is also involved in negative regulation of the pathway. The secreted Wnt modulator, Wise, interacts with LRP,62 although it is not known what the consequence of this binding is at this time. It is interesting to note that Wise and SOST bear homology to each other (see below).62 Both suppression and enhancement of Wnt signaling as the consequences of Wise activity have been reported.62 In the context of LRP and bone growth, the interactions between LRPs and the secreted Wnt antagonist, Dickkopf (Dkk)63 are of special interest. Dkk interferes with canonical Wnt signaling in vertebrates by binding directly to LRP5/6.33, 64, 65 At the same time, Dkk interacts with another transmembrane protein, Kremen (Krm).66 This complex is internalized, thus removing LRP from the cell surface and making LRP unavailable for Wnt signaling.65, 66 The point mutation in the extracellular YWTD motif of LRP5, identified in human patients exhibiting a high bone mass defect,3, 4 prevents binding of Dkk, suggesting that the high bone density in these patients is caused by unrestrained Wnt signaling, not counteracted by Dkk activity.4 These are important findings that suggest that interfering with Wnt signaling may contribute to the clinical management of bone disease. The finding that LRP5 mutations lead to abnormalities in bone growth in humans has led to attempts to generate animal models for bone defects. Two groups have made mice lacking LRP5 function. Fujino et al.67 have provided evidence that Lrp5 deficiency leads to increased plasma cholesterol levels in mice that are fed on a high-fat diet, implying that Lrp5 is also required for normal cholesterol and glucose metabolism. Using a different Lrp5 mutant mouse, however, Kato et al. emphasize that Lrp5 is required for normal proliferation and function of osteoblasts. They also found that Lrp5-deficient mice display persistent embryonic eye vascularization because of a failure of macrophage-induced endothelial cell apoptosis.68 The role of LRP5 in bone mass accrual suggests that Wnt proteins have a role beyond embryonic skeletal patterning in postnatal bone maintenance. It is not clear whether the seemingly contrasting phenotypes described by these two groups can be reconciled. Mouse embryos homozygous for a nonfunctional allele of Lrp6 die at birth and exhibit various developmental abnormalities, including a truncation of the axial skeleton, deletion of the caudal midbrain, and limb defects.49 The defects in Lrp6 mutant mice are a striking composite of those caused by mutations in individual Wnt genes, suggesting a broad role for Lrp6 in the transduction of multiple Wnt signals.49 However, not all known Wnt phenotypes are present in LRP6 mutants. Because the closely related Lrp5 gene is expressed widely in mid-gestation embryos, genetic redundancy is possible, but remains to be tested. The gain of function phenotype associated with a point mutation in the extracellular YWTD motif of LRP5, identified in human patients with high bone mass, is recapitulated in transgenic mice expressing this very same mutant version of LRP5.69 These LRP5 G171V transgenic mice have increased BMD, trabecular thickness, trabecular number, strength, and resistance to fracture. Babij et also reported decreased osteoblast and which is with increased activity of the Wnt signaling pathway in other Recent from and colleagues at in with colleagues at and and has shown a key role for signaling in in bone and These have studied bone formation in response to with a They have shown that from the LRP5 G171V transgenic mice have a more bone formation response to and that the level of required to induce a bone formation response is in the LRP5 G171V transgenic mice. expression have shown in response to the expression of downstream gene of the Wnt signaling pathway are in the LRP5 G171V transgenic mouse the increases observed in mice associated with the LRP5 G171V mutation was observed in that osteoprotegerin levels were also in these mice any in RANKL levels in response to increased to a functional protein level result in osteoclastogenesis. 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