of bone-related diseases

Wnts are cysteine-rich secreted proteins involved in a wide range of developmental processes such as embryonic axis specification and organogenesis [Wodarz, 1998]. Wnts appear to activate a variety of signaling pathways both in vertebrates and in invertebrates. In the so- called « canonical » Wnt/β-catenin pathway, the interaction between Wnt and frizzled receptors leads to inactivation of the kinase GSK-3β. Genetic epistasis experiments suggest that disheveled lies upstream and represses the activity of GSK-3β. As a consequence, β-catenin is stabilized in the cytoplasm and then forms a complex with TCF/LEF to activate transcription of target genes. Frizzled proteins have been shown to function as Wnt receptors [Bhanot, 1996] and they constitute a large family of seven transmembrane receptors with at least ten members in mammals [Fredriksson, 2003;Bhanot, 1996]. All frizzled receptors have a conserved extracellular cysteine-rich domain (CRD) followed by seven putative transmembrane segments. Their cytoplasmic regions differ in length and sequence. Functional analyses in Drosophila and Xenopus embryos indicate that frizzled proteins have distinct functions in Wnt/β-catenin signaling [Sheldahl, 1999]. More recently, members of the low-density lipoprotein receptor-related protein family (LRP) have been shown to be coreceptors for Wnt ligands [Mao, 2001 ; Mao, 2001 ;Pinson, 2000;Tamai, 2000;Wehrli, 2000]. The two closely related proteins LRP5 and LRP6 are single-pass transmembrane receptors that associate with frizzled receptors in a Wnt dependent manner. There is an increasing body of evidence showing that LRP5/6 are required for Wnt/β-catenin signaling. Beside Wnts, dickkopf proteins (Dkk), mainly Dkk1 , Dkk2, and Dkk4, were also shown to bind with high affinity to LRP5/6 and to inhibit Wnt canonical signaling [Semenov, 2001 ;Bafico, 2001 ;Mao, 2001 ;Glinka, 1998]. Despite the fact that modulating LRP5/6 receptor activity by extracellular ligands like Wnts or Dkks leads respectively to the activation or inhibition of /?-catenin signaling pathway, it is unclear how it triggers the signaling cascade in the intracellular compartment. Although it has been previously demonstrated that LRP5/6 interacts with Axin, this interaction only in part explains the mechanism by which this receptor regulates the /?-catenin pathway.

Legend to the Figures

Figure 1 : Fratl interacts with LRP5. COS-7 cells were co-transfected with Frat1-Flag and either LRP5tail-Myc, LRP5tailΔ28-Myc or LRP5tailΔ78- Myc. Control were carried out by transfecting each of the expression construct alone 18 hours after transfection media was replaced by fresh culture media and cultured for additional 24 hours, lmmunoprecipitation (IP) was performed on total cell lysates using anti-Myc antibody. Total cell lysates and immunoprecipitates were analyzed by Western blotting using either anti-Flag or anti-Myc antibody. Arrows indicate the expected band size.

Figure 2 effect of LRP5C truncated mutant forms on Fratl activity. (A) COS-7 cells were transiently co-transfected with TCF-1 expression construct, TOPflash, pTK-Renilla. Where indicated, Fratl expressing vector was added with either empty vector (Vector), LRP5C, LRP5Δ28, LRP5Δ47 or LRP5Δ78. Control experiments were carried out by performing transfection with each of the used constructs alone. 18 hours after transfection media was replaced by fresh culture media and 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ± S. D. from one representative experiment are presented (p<0.01). (B) COS-7 cells were transfected with control vector (I), LRP5C (II) LRP5Δ78 (III), Frati (IV), LRP5C and Frati (V) or LRP5Δ78 and Frati (Vl). 18 hours after transfection media was replaced by fresh culture media and cultured for additional 24 hours. Cells were immuno-stained for Frati expression using a mouse anti-Flag antibody and revealed by a goat anti-mouse antibody conjugated to rhodamine (red fluorescence). Fluorescent cells were visualized under confocal microscopy. Experiment was repeated three times and photos from one representative experiment are shown.

Figure 3. LRP5 expression is necessary for Frati activity. Primary fiboblastics cells from wild type mouse (Irp5y+), heterozygote (Irpδ^, black box) or homozygote (Irp5y) were transiently co-transfected with TCF-1 expression construct, TOPf lash, pTK-Renilla. Empty control vector or Frati expression construct was added to the transfection mix. 18 hours after transfection media was replaced by fresh culture media. Cells tranfected with empty vector were either left untreated (CTRL) or treated with Wnt3a-CM (Wnt3a). 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ± S. D. from one representative experiment are presented (p<0.01 ).

Figure 4. Frati is required for LRP5C activity. (A) COS-7 cells were transiently cotransfected with TCF-1 expression construct, TOPflash, pTK- Renilla. Where indicated either empty vector (Vector), LRP5C, Frati , FratiΔN, LRP5C and Frati or LRP5C and FratiΔN expression constrcuts were added to the transfection mix. 18 hours after transfection media was replaced by fresh culture media and 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ± S. D. from one representative experiment are presented (p<0.01). (B) Cells were co¬ transfected with Frat1-Flag and LRP5C-Myc expression constructs. Control cells were left untransfected (CTRL). Expression of Frat1-Flag or LRP5C-Myc was verified on total cell lysates Western blotting with indicated either anti-Flag or anti-Myc antibody, respectively, lmmunoprecipitation (IP) on total cell lysates using an anti-Myc antibody was performed, lmmunoprecipitates were analyzed by Western blotting with anti-Flag antibody.

Figure 5. Cellular localization of Frati . (A) C3H10T1/2 cells were transfected with Frat1-Flag tagged then either left untreated (I) or treated with Wnt3a-CM (II). C3H10T1/2 cells were transfected with Frat1-Flag and one of the following expression constructs: LRP5C (III), LRP5CΔ28 (IV), LRP5CΔ36 (V), LRP5CΔ47 (Vl) or LRP5CΔ78(VI). Frati localization was immunodetected by mouse anti-Flag antibody and revealed by a goat anti- mouse antibody conjugated to rhodamine (red fluorescence). This assay was performed three time and representative photos are shown. (B) The percentage (average of three experiments) of Frati localized at the membrane, in the cytoplasm or in the nucleus.

Figure 6. Effect of disheveled dominant negative mutant on Frat1/LRP5C activity and association. (A) C3H10T1/2 cells were transfected with control vector, Frati , LRP5C, LRP5CΔ28 or LRP5CΔ78 in the presence or absence of in absence or in presence of disheveled dominant negative (Xdd). Transfection mix systematically included TCF1 plasmid. When indicated cells were treated with Wnt3a-CM (Wnt3a). (B) Cells were cotransfected with Frat1-Flag and LRP5 or LRP5C-Myc in absence or presence of Xdd expression construct, lmmunoprecipitation (IP) on total cell lysates using either anti-LRP5 or anti-Myc antibody was performed, lmmunoprecipitates were analyzed by Western blotting with indicated antibodies. Figure 7. Effect of axin overexpression on Frat1/LRP5 interaction. (A) COS-7 cells were transiently co-transfected with TCF-1 expression construct, TOPflash, pTK-Renilla. Where indicated either empty vector (Vector), Wnt3a, Frati , LRP5C or Frati and LRP5C expression constrcuts were added to the transfection mix. 18 hours after transfection media was replaced by fresh culture media and 24h later luciferase activity was determined in cell lysates and normalized to renilla signal. All experiments were performed in triplicate and repeated three times. Data ± S. D. from one representative experiment are presented (* p<0.01). (B) Cos-7 cells were transiently transfected with Axin-Myc expression vector with either Frat1-Myc, LRP5C-GFP or both expression constructs. Cells lysates were immunoprecipitated (IP) with an anti-Flag antibody (left lanes) or an anti- GFP antibody (right lanes), lmmunoprecipitates were analyzed by Western blotting using anti-GFP, anti-Myc or anti-Flag antibodies. In addition, total cell lysates were analyzed by Western blotting using anti-Myc antibody (lower box). Arrows indicate the size of expected band.

Figure 8. A conceptual model for Wnt signalling through LRP5 receptor. Following activation by Wnt, conformational modification of the LRP5 cytoplasmic domain occurs allowing the recruitment of Frati to the membrane. The activated status of LRP5 receptor is also able to translocate Axin to the membrane where it interacts with Frati . Axin recruitment to the membrane prevents it from enhancing the degradation of β-catenin. In the absence of Wnts, Axin is found within a protein complex that includes APC, GSK3β and β-catenin. The recruitment of Axin by LRP5 in the presence of Wnts will bring GSK3β close to Frati . This allows Frat1/GSK3β interaction and, thus, inhibition of GSK3β by Frati , leading to the stabilization of β-catenin. LRP6, a close homologue of LRP5, probably acts through a similar mechanism. Description of the preferred embodiments The term "disease" means herein an alteration of the health of a mammal, due to internal and/or external causes, said alteration becoming apparent through symptoms and resulting in an impairment of one or more biological functions, such as metabolic functions, and/or in one or more lesions in said mammal. By the term "disorder", it is meant herein a pathological modification of an organ or of a physical or psychological function in a mammal. For the purpose of the invention, the terms "alteration", "impairment", and "modification" as recited above are synonymous. Moreover, the terms "disease" and "disorder" are used herein interchangeably, unless otherwise specified. In the context of the present invention, the expression "bone-related disease" refers to a disorder directly or indirectly affecting bone cells, that gives rise to a condition of clinical relevance for skeletal health. The mechanisms that give rise to such a disease are diverse and may be mediated by primary pathology affecting bone cells (an example is Paget's disease of bone), or indirectly. Indirect mechanisms include the effects of abnormal endocrine secretion of major calcium and skeletal regulating hormones, including sex hormones (estrogen, androgen, progesterone, and the like). Examples include post-menopausal osteoporosis, primary hyper parathyroid ism and Cushing's disease. Bone disease may also arise from the local or systemic effects of cytokines such as in multiple myeloma, periodontal disease. Intrinsic bone disease may be genetic (e.g., epiphyseal dysplasia) or acquired (e.g., osteomyelitis). However, in practice, as knowledge of pathophysiology advances, the distinction between intrinsic and metabolic bone diseases becomes increasingly blurred. Moreover, importantly, pathophysiology of bone disease may also involve target tissues other than bone. An illustrative example is vitamin D deficiency which gives rise to osteomalacia in adults or rickets in childhood. In this respect, the expression "bone-related disease" encompasses at least disorders of mineral metabolism, disorders of parathyroid hormone (PTH) secretion and/or activity, metabolic bone disorders comprising osteoporosis, vitamin D-related disorders, renal bone diseases, hypophosphatasia, dysplastic disorders, infiltrative disorders, extra- skeletal calcification and ossification, miscellaneous disorders, and the like (for a literature reference, see Baron et a/.). By "disorders of mineral metabolism", it is meant herein at least hypercalcaemia of diverse causes, hypocalcaemia of diverse causes, hyperphosphataemia, hypophosphataemia, hypermagnesaemia, hypomagnesaemia, and the like. Under the expression "disorders of PTH secretion and/or activity" are included for instance hyperparathyroidism, hypoparathyroidism, pseudohypoparathyroidism, and the like. "Miscellaneous disorders" encompass at least medullary carcinoma, skeletal toxicity syndromes (e.g., aluminium, iron>cadmium, fluorosis), alveolar bone resorption, non-union and fracture repair, bone reconstruction, ischaemic disorders, osteonecrosis, and the like. A "metabolic bone disorder" includes at least osteoporosis, which may be for instance postmenauposal, involutional, secondary; as well as hypo-remodelling syndromes; and the like. As used herein, the expression "vitamin D-related disorders" relates at least to nutritional, resistance, secondary hyperparathyroidism, ectopic 1-alpha-hydroxylase activity, oncogenic, and the like. By "renal bone disease", it is meant for instance osteitis fibrosa, osteomalacia, osteosclerosis, osteoporosis, adynamic bone disease, and the like. "Hypophosphatasia" refers to, for example, hyperphosphatasia, Paget's disease, Engelman's disease, and the like. "Dysplastic disorders" may be for instance sclerosing bone dysplasias and osteoporosis, fibrous dysplasia, mucopolysaccharidoses, periostoses, ankylosing spondylarthritis, osteochondroses, osteophytosis, Diffuse Osteopathic Skeletal Hyperostosis (DISH), osteogenesis imperfecta, genetic disorders, and the like. "Infiltrative disorders" include at least primary skeletal neoplasms, secondary skeletal neoplasms, systemic mastocytosis and histiocytosis, sarcoidosis, oxalosis, and the like. "Extra-skeletal calcification and ossification" may be for example renal bone disease, fibrodysplasia ossificans progressiva, nephrolithiasis, and the like. In an embodiment of the present invention, a "bone-related disease" is osteoporosis. According to the invention, the term "mammals" encompasses animals and humans. In an embodiment, a "mammal" is a human. A "compound" herein refers to any type of molecule, biological or chemical, natural, recombinant or synthetic. For instance, such a compound may be a nucleic acid (e.g., an antisense or sense oligonucleotide including an antisense RNA), a protein, a fatty acid, an antibody, a polysaccharide, a steroid, a purine, a pyrimidine, an organic molecule, a chemical moiety, and the like. The term "compound" is preferably used herein to refer to a compound which exhibits the function of interest, i.e., the ability to modulate Frat-LRP interaction. In this respect, also encompassed by the term "compound" are fragments, derivatives, structural analogs, and combinations thereof, all of them being functional, i.e., being capable of modulating Frat-LRP interaction. As used herein, a "molecule" is of any type, biological or chemical, natural, recombinant or synthetic. For instance, such a molecule may be a nucleic acid (e.g., an antisense or sense oligonucleotide including an antisense RNA), a protein, a fatty acid, an antibody, a polysaccharide, a steroid, a purine, a pyrimidine, an organic molecule, a chemical moiety, and the like. The terms "molecule" and "compound" thus refer to the same structures. However, as used herein, these terms are not equivalent, since a "compound" is, as defined above, capable of modulating Frat-LRP interaction, whereas a "molecule" either displays a biological function, which is thus different than the ability to modulate Frat-LRP interaction, or it is inert, i.e., it does not have any biological function. As used herein, the terms "activity" and "active", and "function" and "functional" are synonymous, respectively. Moreover, the terms and expressions "biological activity", "biological function", "activity", and "function" are also synonymous. The term "Frat" herein refers to all Frat (for frequently rearranged in advanced T-cell lymphomas) proteins and homologs thereto, preferably from mammals. The term "Frat" may, depending on the context, also refer to the nucleic acids corresponding to all Frat proteins and homologs thereto. This definition includes, for instance, Frati (Jonkers et al., 1997), Frat2 (Saitoh et al., 2001 ; Freemantle et al., 2002), and Frat3 (Jonkers et al., 1999), as well as functional (i.e., capable of behaving and acting like Frat) fragments and derivatives thereof, such as the FRATtide peptide (Thomas et al., 1999). As used herein, the term "LRP" encompasses all LRPs (for |ow- density lipoprotein-receptor-related p/oteins) and homologs thereto, preferably from mammals. The term "LRP" may, depending on the context, also refer to the nucleic acids corresponding to all LRP proteins and homologs thereto. This definition includes, for instance, LRP5 (Kim et al., 1998; Hey et al., 1998) and LRP6 (Brown et al., 1998; Tamai et al., 2000; Pinson et al., 2000), as well as functional (i.e., capable of behaving and acting like LRP) fragments and derivatives thereof. A "fragment" include a part of a Frat or a LRP of reference, which retains the biological activity of said Frat or LRP of reference. A functional "derivative" of a Frat or LRP of reference may be, for example and without any limitation, a protein, a peptide, an hormone, an antibody, either natural or synthetic, as soon as it may be derived structurally from said Frat or LRP of reference and it exhibits the biological function thereof. The expression "Frat-LRP interaction" refers not only to the structural cooperation between the two partners Frat and LRP, but also the functional cooperation therebetween, as illustrated in the Examples. By "modulating Frat-LRP interaction", it is meant that said interaction is either positively or negatively affected by acting directly or indirectly on the structural and/or functional cooperation between Frat and LRP. When the interaction is "positively affected", it is meant that the interaction is induced, promoted, activated, enhanced, increased. On the contrary, when the interaction is "negatively affected", it is meant that the interaction is suppressed, inhibited, reduced, decreased. For example, the following modulating effects are encompassed by this definition: - stabilizing (positive) or destabilizing (negative) one partner (Frat or LRP), both partners (Frat and LRP), or the two-partner complex; - activating (positive) or inhibiting (negative) inducers of one or both Frat and LRP, or of the complex; - inhibiting (positive) or activating (negative) inhibitors of one or both Frat and LRP1 or of the complex; - increasing (positive) or decreasing (negative) the expression and/or the cell turnover and/or the availability of one or both Frat and LRP so that the two-partner complex cannot be readily formed. As used herein, a "pharmaceutical composition" is equivalent to a "pharmaceutical preparation", both referring to a "drug" as commonly understood by the skilled artisan in the field of the invention. More precisely, said "pharmaceutical composition" or "pharmaceutical preparation" or "drug" comprises a pharmaceutically acceptable amount of one or more compounds and, optionally, one or more molecules, all of them being generally associated to, or contained in, at least one pharmaceutically acceptable carrier. The "pharmaceutically effective amount" of an active compound is the amount of said compound that results in amelioration of symptoms in a mammal. A "pharmaceutically acceptable carrier", also referred to as an "adjuvant", is conventional and may easily be chosen by the one skilled in the art, depending on the administration route of the drug under consideration, by relying on the general knowledge in techniques for formulating drugs (see the Remington reference). According to a first aspect, the present invention relates to a method for preventing and/or treating a bone-related disease in a mammal in need of such treatment, wherein said method comprises: administering to said mammal an effective amount of a pharmaceutical composition comprising at least one compound capable of modulating Frat-LRP interaction, so that GSK-3 is inhibited and, thus, β-catenin is stabilized, i.e., it is not submitted to phosphorylation by GSK-3. Such a pharmaceutical composition comprises one or more different compounds and, optionally, one or more molecules which, as defined above, do not exhibit the ability to modulate Frat-LRP interaction. For instance, these molecules may only act as adjuvants or carriers, such as polylactic acid, polyglycolic acid, polydioxanone, collagen, albumin, detergent (e.g., polyoxyethylenesorbitan), and the like. Other useful molecules may have a biological function (hereafter referred to as "biologically-active molecules"), different that the one of compounds capable of modulating Frat-LRP interaction, but the association of which may be of interest regarding bone formation and protection. In this respect, biologically-active molecules may be vitamins. Other useful biologically-active molecules may be molecules that promote tissue growth or infiltration, including bone morphogenic proteins such those described in U.S. Patent No. 4,761 ,471 and PCT Publication WO 90/11366, osteogenin (Sampath et al., 1987), and NaF (Tencer et al., 1989). Yet other biologically-active molecules may be targeting molecules, i.e., molecules that bind to (have affinity with) the tissue of interest. Examples of bone-targeting molecules include tetracyclines; calcein; biphosphonates; polyaspartic acid; polyglutamic acid; aminophosphosugars; peptides known to be associated with the mineral phase of bone such as osteonectin, bone sialoprotein and osteopontin; bone specific antibodies; proteins with bone mineral binding domains; and the like (for example, see Bentz et al. in EP 0512844 and Murakami et al. in EP 0341961 ). For the purpose of determining the pharmaceutically effective amount of compounds as defined above, toxicity and therapeutic efficacy of said compounds can be determined by standard pharmaceutical procedures in cell cultures (in vitro) or in experimental animals (in vivo). For example, the LD50 (the dose lethal to 50% of the population), as well as the ED50 (the dose therapeutically effective in 50% of the population) can be determined using methods known in the art. Accordingly, the data obtained from cell culture assays (in vitro) and/or animal model studies (in vivo) can be used in formulating a range of dosage of these compounds which lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. In the context of the invention, administration of a drug may be performed via any route such as locally, orally, systemically, intravenously, intramuscularly, mucosally, using a patch, using encapsulating or embedding liposomes, microparticles, microcapsules, and the like. According to a second aspect, the present invention is related to a method for selecting a compound useful for preventing and/or treating a bone-related disease in a mammal in need of such treatment, wherein said method comprises: a) testing the ability of a candidate compound to modulate Frat-LRP interaction in vitro and/or in vivo; and b) if said candidate compound modulates Frat-LRP interaction, selecting said compound. In an embodiment, this method further comprises purifying the selected compound. Methods for detecting a modulating effect on Frat-LRP interaction include both in vitro and in vivo procedures (e.g., protein-protein binding assays, biochemical screening assays, immunoassays, cell-based assays, animal model experiments, which are well-characterized in the art). For instance, the person skilled in the art may use only one in vitro and/or one in vivo selection technique. However, in order to strengthen the validity and reproducibility of the results, this person may prefer to use at least two in vitro and/or at least two in vivo selection methods. Appropriate examples of conventional procedures for showing a modulating effect on Frat-LRP interaction are FRET, BRET, any method for detecting an energy transfer between the Frat and LRP, double-hybrid assays, ELISA- like methods, and the like... The present invention also encompasses: - the compounds themselves and/or for use as medicaments ; and - the use of at least one compound as defined above for the manufacture of a pharmaceutical composition for preventing and/or treating bone-related diseases in a mammal. Summary of the Examples

LDL receptor-related protein 5 (LRP5) has been identified as a Wnt co receptor involved in the activation of the /?-catenin signaling pathway. To improve our understanding of the molecular mechanisms by which LRP5 triggers the canonical Wnt signaling cascade, we have screened for potential partners of LRP5 using the yeast two-hybrid system and identified Frati as a protein interacting with the cytoplasmic domain of LRP5. We demonstrate here that LRP5/Frat1 interaction is required for the yff-catenin nuclear translocation and TCF1 transcriptional activation. Addition of Wnt3a or overexpression of constitutively active truncated LRP5 induces Frati recruitment to the cell membrane. Overexpression of a dominant negative form of dishevelled (DvI) shows that this protein positively affects LRP5/Frat1. Furthermore, the fact that dominant negative DvI does not interfere with LRP5C/Frat1 interaction can explain how LRP5C is capable of acting independently of this major Wnt signaling player. Axin that has been shown to interact with LRP5 and to be recruited to the membrane through this interaction has been found to co- immunoprecipitate with Frati and LRP5. We propose that recruitment of Axin and Frati to the membrane by LRP5 leads to both Axin degradation and Frati -mediated inhibition of GSK3. As a consequence, β-catenin is no longer bound to Axin or phosphorylated by GSK-3, resulting in TCF1 activation.

Examples

I - Materials and Methods Yeast two-hybrid screening The MATCHMAKER yeast two-hybrid (Y2H) system and the mouse 11- day and 19-day embryo cDNA libraries were purchased from Clonetech. The bait for library screening was the intracellular domain of LRP5 (1419- 1615 residues). Y2H screening was carried out as suggested by the manufacturer. The interaction of the target proteins was determined phenotypically by growing of yeast clones on His-deficient media and measuring β-galactosidase activity. First confirmation for interaction of target proteins was done as follow, both bait and prey constructs were retrieved from yeast clone of interest (as described by the manufacturer), re-introduced again in the same yeast strain and confirmed for their ability to grow on His-deficient media and to induce β-galactosidase activity. After this first challenge, interaction was further confirmed as follow, bait and prey cDNAs were removed from their original plasmid and recloned using appropriate restriction sites in prey and bait plasmids, respectively, re¬ introduced again in the same yeast strain and confirmed for their ability to grow on His-deficient media and to induce β-galactosidase activity. A potential target will be considered for further analysis if passed those two assays.

Wnt3a-conditioned media preparation Wnt3a-conditioned media (Wnt3a-CM) was prepared as described by Shibamoto et al. [Shibamoto, 1998]. Briefly, to collect the conditioned medium from cultures of Wnt-3aproducing L cells, we seeded these cells were seeded at a density of 6x10 6 cells in a 125 cm2 flask containing DMEM with 10% FCS. 24 hours after seeding, medium was changed to DMEM with 2% FCS and cultured them for 3 days. Then Wnt3a-CM was harvested, centrifuged at 1000 g for 10 min, and filtered through a nitrocellulose membrane. The activity of Wnt3a-CM was assayed on normal L cells by examining increase in /?-catenin as described by Willert et al. [Willert, 1999]. Wnt3a-CM was added to cells at 20% final concentration in all subsequent experiments.

Plasmids and constructs Myc-tagged LRP5 was described previously [Gong, 2001], derived truncated constructs were amplified by PCR and fused to the indicated tag-epitope into pcDNA3.1 vector. /?-catenin, disheveled and Wnt3a cDNAs were isolated by RT-PCR and confirmed by nucleotide sequencing. Isolated sequences were tagged with appropriate epitope and subcloned into the expression plasmid pcDNA3.1. Dominant negative form of dishevelled (Xdd) was generated as previously described [Sokol, 1996; Yanagawa, 1995]. Stable mutant /?-catenin (/?-catenin*) was generated as described by Morin et al. [Morin, 1997]. Axin expression vector was kindly provided Dr. DL Shi (CNRS, France). Flag-Frat1 expression construct was kindly provided by Dr. E. Fraser (Institute of cancer research, UK) and dominant negative form, Frati N, was generated as described by Li et al. [Li, 1999]. The expression of all tagged expression constructs was confirmed by Western blotting.

Ce// Cultures and Transfection COS-7 monkey kidney cells (ATCC) and, mouse fibroblast L cells (ATCC) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Mesenchymal mouse C3H10T1/2 cells were grown in σ-modified Eagle's medium supplemented with 10% FBS. Cells were plated in 24-well plates at 4x10 cells/well 24h prior to transfection, then transiently transfected with the indicated construct (1 μg total DNA) using DNA-lipid complex Fugene 6 (Boehringer Mannheim) according to the manufacturer's protocol. 16 h after transfection, cells were washed, and cultured in medium at 2% fetal calf serum for an additional 48h under indicated conditions. Cells were then collected for luciferase assays, immunoprecipitation or Western blotting. Controls were carried out by replacing expression constructs by empty vector. When the luciferase reporter construct was used, to assess transfection efficacy, 20 ng of pRL-TK (Promega) encoding a Renilla luciferase gene downstream of a minimal HSV-TK promoter was systematically added to the transfection mix. Luciferase assays were performed with the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions. 10 μl of cell lysate was assayed first for firefly luciferase and then for Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.

Western blot and immunoprecipitation assay For Western analysis and immunoprecipitation (IP), transiently transfected cells were harvested and lysed with M-PER Mammalian Protein Extraction Reagent (Pierce). Total protein content in cell lysates was estimated using the BCA-Protein assay (Pierce). To control for protein expression, 10μg of total protein were loaded on Nu-PAGE 4-12% (Invitrogen). After electrophoresis, proteins were transferred on Hybond-ECL nitrocellulose membrane (Amersham-biosciences) and revealed with either HRP anti-flag (monoclonal mouse anti body, Sigma), HRP anti-c- Myc (Boehringer Mannheim) or HRP anti-GFP (polyclonal goat anti body, Abeam). Immunoreactive proteins were visualized by enhanced chemiluminescence detection according to the manufacturer (ECL+ Amersham-biosciences). IP was the carried out on 500μg of total protein. Total protein were incubated with 50μl of μMacsprotein G microbeads (Miltenyi Biotec) and 2μg of indicated anti-epitope anti-body at room temperature for 1h. The magnetically labeled immune complex is passed over a microcolumn placed in magnetic field. This complex is bound to the column where other proteins are washed-away. lmmunoprecipitated proteins are eluted from the microcolumn with hot denaturant gel loading buffer, then subjected to Western blotting as described above.

Confocal microscopy assay C3H10T1/2T1/2 cells are plated at 40 000 cell per wells in 6 wells plate, each well contain a sterile micro cover glass which can bee removed for observation. 48 hours after transfection or treatment, cell are fixed with 3.7% formaldehyde (sigma) for 10 minute, watched tow time with PBS. Cell are also permeabilized by PBS/0.025% triton 100x (sigma) for 5minut.es, and blocked in PBS/3% BSA for 15minutes. After that Cells are incubated with the primary antibody, a Mouse monoclonal IgGI (Santa Cruz) against Flag tag and a rabbit poly clonal anti Cmyc (Santa Cruz) at the concentration of 5μg/ml in PBS, over night at 4°c. After being washed tow time cells are incubated with the secondary anti body an Goat anti rabbit conjugated to FITC (Santa Cruz 2012) and Goat anti mouse conjugated to Rhodamine (Santa Cruz sc2092) at 1/100 for an hour at room temperature. The cover glass are washed tree time and mounted on glass slide and viewed with a confocal laser-scanning microscope (LSM510, Carl-Zies, Germany).

Derivation of fibroblastics cells from KO Irp5 mouse We using calvarias from Irpδ KO, heterozygote or wild type mouse, 1 at 3 days old. Calvarias were collected in 15 ml tube and washed tow time with Phosphate buffered saline with 0.1 % of Penicilline/Streptomycine. Calvarias were treated with collagenase type IA (sigma) and dyspase type Il (Boehringer) σMEM medium (Gibco) for 1 minutes at 37° c under agitation. After that supernatant was discarded and this operation was performed tree times. Cells are collected in σMEM medium with 10% of fetal cow serum (HiClone) and centrifuged at 1500 rpm for 10 minutes. Supernatant was discarded and cells resuspended in 10 % FCS in αMEM medium, than plated. Fibroblastic cells are transfected in the same way described upper.

Il - Results LRP5 interacts with Frati It has been clearly established that LRP5/6 receptor is required for Wnt to induce the canonical β-catenin signaling pathway. Interestingly, in the absence of Wnt, overexpression of LRP5 lacking either the extracellular or both the transmembrane and extracellular domains leads to the activation of the canonical signaling pathway (data not shown, [Mao, 2001]). These data clearly show that the cytoplasmic tail of LRP5 is able to interact with intracellular players resulting in the activation of Wnt pathway. We have therefore performed a Y2H screen to identify proteins that interact with the LRP5 cytoplasmic domain. LRP5 cytoplasmic domain was used as bait to screen for potential partners in mouse embryo cDNA libraries (11 and 19 days). From each library, around 20 million clones were screened; clones showing interaction with LRP5 bait in the primary screen were confirmed in secondary screen (see material and methods). Prey from confirmed clones were selected for the specificity of interaction with LRP5 cytoplasmic tail by testing for interaction with two other unrelated baits (i.e., Id3 and Sprouty2). In summary, 35 and 77 clones were confirmed respectively from the 11 -day and the 19-day embryo cDNA library. Preys from confirmed clones were sequenced to identify the corresponding cDNA. After sequence analysis, we ended with 45 distinct cDNAs, from which 85% were found in both libraries. Among them we identified "frequently rearranged in advanced T-cell lymphoma 1" (Frati) as a new potential partner for LRP5 and further investigated the physiological significance of LRP5-Frat1 interaction. LRP5 cytoplasmic domain (LRPδtail) and murine Frati were cloned in expression vectors as Myc or Flag-tagged, respectively, and expression in COS-7 cells was verified by Western blotting (Fig. 1). To confirm interaction identified in Y2H, Myc-LRP5tail and flag-Frat were co- expressed in COS-7 cells and found to co-immunoprecipitate (Fig. 1 ). In addition, Flag- Frati was shown to co-immunoprecipitate with the Myc- LRP5 full length protein (figure 1 ). Co-expression of Flag-Frat1 with two other unrelated Myc-tagged proteins (i.e., Id3, Sprouty2) did not allow co- immunoprecipitation (data not show). To further investigate the domain of LRP5 cytoplasmic tail involved in this interaction, we generated truncated LRPδtail Myc-tag constructs deleted of 28, 36, 47 or 78 C-terminal residues, respectively named LRP5tailΔ28, LRP5tailΔ36 LRP5tailΔ47 and LRP5tailΔ78. These constructs were assessed for interaction with Frati by co-IP assay and data clearly show that Frati interacts with either LRP5tailΔ28 or LRP5tailΔ36 (Fig. 1), but not with LRP5tailΔ47 and LRP5tailΔ78 (data not shown). Altogether our data indicate that Frati specifically interacts with LRP5 cytoplasmic domain raising the question of the biological significance of this interaction and its potential role in the activity of both LRP5 and Frati .

LRP5 and Fratl interaction is necessary for Wnt signaling LRP5 lacking the extra cellular domain, LRP5C, acts as a constitutive active receptor and triggers /?-catenin pathway in the absence of Wnt proteins (Fig.2A and [Mao, 2001]). LRP5C deleted of its last 78 amino acids (LRP5CΔ78) failed to activate the TCF transcriptional activity whereas LRP5CΔ28 was as active as LRP5C. LRP5CΔ47 displays some activity but significantly minor than LRP5C (Fig. 2A). Then, we tested the effect of overexpression of Fratl on the activity of LRP5C and its derivative mutants. Overexpression of Fratl alone is able to induce TCF 1 activation (Fig.2A, and [Li, 1999]). As depicted in Fig. 2A, an additive effect was found when LRP5C and Fratl were overexpressed together. LRP5CΔ28 did not cooperate with Fratl to induce TCF1 activation, and surprisingly Fratl activity was found to be inhibited by LRP5CΔ78 (Fig. 2A). In concordance with the transcriptional analysis, overexpression of either LRP5C or Fratl induced /?-catenin nuclear translocation (Fig. 2B panel Il and IV). LRP5CΔ78 had no effect on /?-catenin localization (Fig.2B, panel III), but it completely blocked Fratl induced-/?-catenin nuclear translocation (Fig.2B, panel XVI). These data strongly suggest that LRP5 is required for Frati activity. To further support these findings, we prepared primary fibroblastic cells from wild-type Lrp5+'\ heterozygous Lrp5H- and homozygous Lrpδ1- mice. As shown in figure 3, Wnt3a was able to strongly induce TCF-1 transcriptional activity in primary cells from Lrpδ*'* mice, whereas cells from Lrpδ*1- or Lrpδ'- mice showed little or no response, respectively (Fig. 3). As expected, overexpression of Frati in cells derived from wild type mice (Lrpδ*'*) induced TCF-1 transcriptional activity, whereas it failed to affect this activity in cells derived either from Lrpδ*'- or Lrpδ1- animals (Fig. 3). Having sufficient data supporting that LRP5 is required for Frati to induce canonical Wnt signaling, we next addressed whether Frati is involved in the constitutive activity of LRP5C. To answer this question we made a Frati construct deleted of the N- terminal region (FratiΔN) that has been described to be capable of inhibiting Wnt activity through constitutive binding to GSK3 [Thomas, 1999]. As expected, FratiΔN almost completely inhibited Wnt3a activity and in addition it significantly inhibited LRP5C activity (Fig. 4A). Co-IP assays clearly indicates that FratiΔN is no longer able to interact with LRP5C (Fig. 4B) or LRP5 (data not shown). Furthermore, overexpression of FratiΔN blocked /?-catenin nuclear translocation induced by LRP5C (data not shown). These data demonstrate that LRP5 activity is at least partially dependent on the interaction with Frati .

LRP5 signaling controls Frati cellular localization Frati has been shown to be able to localize into different cell compartments [Franca-Koh, 2002; Freemantle, 2002]. We tested the effect of LRP5C on Frati cell distribution by immunohistochemistry using confocal microscopy. Overexpressed Frati predominantly localized in the cytoplasm, yet a significant fraction (~25%) was also found on the cell membrane (Fig. 5A panel I; Fig. 5B). Stimulation of cells with Wnt3a induced a shift in Frati cell localization from cytoplasm to the membrane, where over 80% of Frati could be localized (Fig. 5A panel II; Fig. 5B). A similar effect was obtained when LRP5C or LRP5CΔ28 were overexpressed in the presence of Frati (Fig. 5A panel III-IV; Fig. 7B). Overexpression of LRP5CΔ47 caused a reduction of Frati levels on the membrane and induced a small but significant translocation of Frati into the nucleus (-10%; Fig. 5B). Surprisingly, LRP5CΔ78, which we have shown to inhibit Frati activity, induces the majority of Frati to be localized into the nucleus (Fig. 5A panel VII). The percentage of membrane, cytoplasm or nucleus location of Frati was determined and data are shown in figure 5B. In summary, data presented herein strongly suggest that the interaction of LRP5 with Frati modulate Wnt/LRP5 signaling by regulating Frati cellular localization.

Dominant negative DvI does not interfere with LRP5C/Frat1 interaction Frati has been shown to interact with several other components of Wnt//S- catenin pathway, including dishevelled (DvI) and GSK3 [Farr, 2000; Li, 1999]. We have investigated how DvI might be involved in LRP5/Frat1 interaction and its ability to transduce signal. Overexpression of Xdd, a DvI construct with only the DEP domain and acting as a dominant negative [Sokol, 1996; Yanagawa, 1995], strongly inhibits TCF-1 transcriptional activity induced by either Wnt3a or Frati , whereas it has no effect on LRP5C or LRP5CΔ28 activity (Fig. 6A). Our results confirm previously reported data demonstrating that LRP5C acts in a Dvl-independent manner [Li, 2002]. We next assessed whether Xdd could interfere in Frati interaction with either LRP5 or LRP5C. As shown in figure 6B, Xdd had no effect on LRP5C and Frati interaction, but it inhibits LRP5/Frat1 interaction as determined by co-IP assay (Fig. 6B). Furthermore, Xdd overexpression did not inhibited LRP5C-induced Frati membrane recruitment whereas it totally blocked wild-type LRP5-mediated Fratl membrane localization (data not shown). Those data suggest that although DvI affects LRP5/Frat1 interaction and subsequent Wnt signaling, it is unable to behave similarly with LRP5 deprived of its extracellular domain, thus explaining the fact that LRP5C is acting independently of this major Wnt signaling player.

LRP5, Fratl, and Axin interact in the same complex Axin is known to play an important role in the regulation of /?-catenin phosphorylation and degradation. Several authors have already demonstrated that Axin directly binds LRP5 and LRP5C [Tolwinski, 2003; Mao, 2001]. Axin overexpression inhibits Wnt3a (data not shown) or Fratl inducing TCF-1 transcriptional activity (Fig. 7A, [Thomas, 1999]). Our data obtained with truncated LRP5 mutants (see Fig. 1) and results reported by Mao et al. [Mao, 2001] suggest that Fratl and axin might interact with LRP5 within close motifs. We have therefore asked whether LRP5, Fratl and axin interact in the same complex or whether Fratl and axin compete for the binding on LRP5. We have performed co-IP assays to address this question. Data shown in figure 7B clearly indicate that axin overexpression does not disrupt LRP5C interaction with Fratl , because all the three proteins could be coimmunoprecipitated within the same complex. As expected, Fratl did not coimmunoprecipitate with axin in the absence of LRP5C (Fig. 7B). Similar data were obtained using LRP5 instead of LRP5C (data not shown). Altogether, these data suggest that LRP5 interact and recruit to the membrane not only axin, as already demonstrated by Mao et al. [Mao, 2001], but also Fratl .

Ill - Discussion Although it has been clearly established that LRP5/6 is a major actor in the activation of the Wnt canonical pathway (for review [Nusse, 2001]), the precise molecular mechanisms by which LRP5/6 participate in this important cascade remain to be elucidated. Currently there are no clear evidences of a binding of any Wnt protein to LRP5/6, and there are only few demonstrations on how LRP5/6 transduce intracellular signal leading to the activation of /?-catenin pathway. In fact, unlike other low-density lipoprotein related receptors (e.g., LDL receptor), the cytoplasmic tail of LRP5 displays no NPXY motif that was identified to interact with the adaptor protein Dab-1 (Gotthardt et al., 2000). The fact that LRP5/6 cytoplasmic tail is rich in proline residues suggests that it might be able to bind SH3-motif-like but nobody has investigated such a possibility. Today the only protein shown to be able to interact with LRP5 cytoplasmic domain is axin [Mao, 2001 ; Tolwinski, 2003]. By performing a Y2H screen in order to identify additional partners for LRP5 receptor, we have identified Frati and we present herein evidences of the crucial role that this interaction plays in the activation of the canonical Wnt//?-catenin cascade. Frati (also named GBP) was first identified in Xenopus as a protein that inhibits GSK-3 in vivo, appearing to act as a positive regulator of the Wnt signalling pathway by stabilising /?-catenin [Yost, 1998]. Further studies performed in Xenopus showed that Frati inhibited GSK-3 activity towards /?-catenin, at least in part, by preventing Axin binding to GSK-3 [Ferkey, 2002; Fraser, 2002; Farr, 2000]. Transfection studies in mammalian cells have confirmed the role of Frati in the stabilisation of /?-catenin and have shown the presence of FRAT1 in complexes with DvI, GSK-3 and Axin [Li, 1999]. Taken together, these findings suggest that Wnt signalling causes a recruitment of Frati into such complexes, leading to Frati -mediated dissociation of GSK-3 from Axin. In the present study we clearly demonstrate that Wnt3a induces the localization of Frati at the membrane through the binding to LRP5 cytoplasmic tail. Interaction of Frati with LRP5 is required to activate the canonical /?-catenin signalling pathway. Mao et al. [Mao, 2001] have shown that Axin interacts with LRP5 and the region necessary for binding Axin is located within the 36 C-terminal amino acids in the LRP5. Results presented herein show that the region required for binding Frati and recruiting it to the membrane was narrowed down to the 47 C-terminal amino acids of LRP5. Our results demonstrate that both Frati and Axin interact with LRP5 cytoplasmic tail but they do so most likely on distinct motifs. These findings strongly suggest that following Wnt stimulation, Frati is recruited to the membrane where it interacts with LRP5 in the same complex as Axin thus enabling it to mediate dissociation of GSK-3 from Axin and subsequent stabilization of /?-catenin (Fig. 8). It remains to be determined whether GSK-3 is indeed involved in this complex. The C- terminus of LRP5, where Frati and Axin binding occurs, contains three repeated motifs (PPT/SP) that might constitute phosphorylation sites for serine/threonine kinases. However, today there is no evidence for such a phosphorylation event. Additional investigations are therefore required to clarify these possibilities.

Our data show that LRP5/Frat1 interaction is required for the /?-catenin signalling. This was clearly demonstrated using fibroblastic cells derived from LRP5 knockout animals. Furthermore, LRP5 truncated of the C- terminal polypeptide involved in the interaction with Frati inhibits Frati- mediated transcriptional activity and, more interestingly, induces the nuclear localization of Frati . In our hands, in the absence of Wnt or LRP5C overexpression, Frati was able to induce TCF1 transcriptional activation and located predominantly in the cytoplasm. From our data one could conclude that nuclear localization of Frati was concomitant with its loss of activity toward TCF1. Franca-Koh et al. [Franca-Koh, 2002] have determined a nuclear export sequence within Frati amino acid sequence and showed that Frati regulates GSKK-3 nuclear export, however the precise role of Frati -mediated GSK3 nuclear export in Wnt signaling has not investigated. The precise mechanism that underlies the constitutive activity LRP5C remains currently unclear. Our data and those from Mao et al. [Mao, 2001] strongly suggest that LRP5C activates the /?-catenin pathway in a DvI independent manner. DvI an important regulator of the Wnt canonical pathway is known to interact with Frati and also Axin to regulate /?-catenin stabilization (Li et al., 1999). Our Co-IP experiments show that a dominant negative form of DvI, that inhibits Wnt induced TCF1 transcriptional activation, is unable to disrupt LRP5C/Frat1 interaction. On the opposite, dominant negative form of DvI inhibits the LRP5/Frat1 interaction. These data demonstrate that DvI is not involved in LRP5C constitutive activity because it is unable to modulate Frati interaction with this truncated form of LRP5. Very recently, Liu et al. [Liu, 2003] have demonstrated that LRP5/6 forms an inactive homomeric complex, whereas constitutively active LRP5/6 mutants (LRP5/6C) were monomeric and whose activity is inhibited by forced dimerization. Their findings demonstrate that Wnt induces conformational switch in the LRP5/6 extracellular domain that relieves allosteric inhibition imposed on the intracellular domain. However, functional and biochemical studies presented herein and by Mao et al. [Mao, 2001] the constitutive activity of LRP5 mutant is not fully comparable to the Wnt activation through wild-type LRP5 receptor. Altogether, our data suggest that conformational alteration of LRP5 intracellular induced by truncating the extracellular domain is slightly distinct from that induced by Wnts.

In summary, based on data presented in this report and observations previously reported, we propose a conceptual model depicted in figure 8 for Wnt signalling through LRP5 receptor. Following Wnt activation, conformational modification of the LRP5 cytoplasmic domain occurs allowing the recruitment of Frati to the membrane. The activated status of LRP5 receptor is also able to translocate Axin to the membrane where it interacts with Frati . Axin recruitment to the membrane prevent it from enhancing the degradation of β-catenin. In the absence of Wnts, Axin is found within a protein complex that includes APC, GSK3β and /?-catenin. Recruiting Axin interaction with LRP5 in the presence of Wnts will bring GSK3b close to Frati and thus allowing Frat1/GSK3/? interaction and consequent inhibition of GSK3/? by Frati leading to the stabilization of /?-catenin. LRP6, a close homologue may be also acting through similar mechanism.

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