title: Bone overgrowth-associated mutations in the LRP4 gene impair sclerostin facilitator function.
abstract: Humans lacking sclerostin display progressive bone overgrowth due to increased bone formation. Although it is well established that sclerostin is an osteocyte-secreted bone formation inhibitor, the underlying molecular mechanisms are not fully elucidated. We identified in tandem affinity purification proteomics screens LRP4 (low density lipoprotein-related protein 4) as a sclerostin interaction partner. Biochemical assays with recombinant proteins confirmed that sclerostin LRP4 interaction is direct. Interestingly, in vitro overexpression and RNAi-mediated knockdown experiments revealed that LRP4 specifically facilitates the previously described inhibitory action of sclerostin on Wnt1/β-catenin signaling. We found the extracellular β-propeller structured domain of LRP4 to be required for this sclerostin facilitator activity. Immunohistochemistry demonstrated that LRP4 protein is present in human and rodent osteoblasts and osteocytes, both presumed target cells of sclerostin action. Silencing of LRP4 by lentivirus-mediated shRNA delivery blocked sclerostin inhibitory action on in vitro bone mineralization. Notably, we identified two mutations in LRP4 (R1170W and W1186S) in patients suffering from bone overgrowth. We found that these mutations impair LRP4 interaction with sclerostin and its concomitant sclerostin facilitator effect. Together these data indicate that the interaction of sclerostin with LRP4 is required to mediate the inhibitory function of sclerostin on bone formation, thus identifying a novel role for LRP4 in bone.
authors.affiliation: Musculoskeletal Disease Area, Novartis Institutes for Biomedical Research, Novartis Pharma AG, Basel, Switzerland. olivier.leupin@novartis.com
authors.forename: Olivier
authors.initials: O
authors.lastname: Leupin
authors.forename: Elke
authors.initials: E
authors.lastname: Piters
authors.forename: Christine
authors.initials: C
authors.lastname: Halleux
authors.forename: Shouih
authors.initials: S
authors.lastname: Hu
authors.forename: Ina
authors.initials: I
authors.lastname: Kramer
authors.forename: Frederic
authors.initials: F
authors.lastname: Morvan
authors.forename: Tewis
authors.initials: T
authors.lastname: Bouwmeester
authors.forename: Markus
authors.initials: M
authors.lastname: Schirle
authors.forename: Manuel
authors.initials: M
authors.lastname: Bueno-Lozano
authors.forename: Feliciano J Ramos
authors.initials: FJ
authors.lastname: Fuentes
authors.forename: Peter H
authors.initials: PH
authors.lastname: Itin
authors.forename: Eveline
authors.initials: E
authors.lastname: Boudin
authors.forename: Fenna
authors.initials: F
authors.lastname: de Freitas
authors.forename: Karen
authors.initials: K
authors.lastname: Jennes
authors.forename: Barbara
authors.initials: B
authors.lastname: Brannetti
authors.forename: Nadine
authors.initials: N
authors.lastname: Charara
authors.forename: Hilmar
authors.initials: H
authors.lastname: Ebersbach
authors.forename: Sabine
authors.initials: S
authors.lastname: Geisse
authors.forename: Chris X
authors.initials: CX
authors.lastname: Lu
authors.forename: Andreas
authors.initials: A
authors.lastname: Bauer
authors.forename: Wim
authors.initials: W
authors.lastname: Van Hul
authors.forename: Michaela
authors.initials: M
authors.lastname: Kneissel
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D019943:Amino Acid Substitution; D000818:Animals; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism*; D005819:Genetic Markers / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D026502:LDL-Receptor Related Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D020125:Mutation, Missense; D010011:Osteocytes / Q000378:metabolism*; D010012:Osteogenesis; D015398:Signal Transduction / Q000235:genetics; D051155:Wnt1 Protein / Q000235:genetics / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics / Q000378:metabolism
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D019485:Bone Morphogenetic Proteins; D005819:Genetic Markers; D026502:LDL-Receptor Related Proteins; C551781:LRP4 protein, human; C422903:SOST protein, human; C495247:WNT1 protein, human; D051155:Wnt1 Protein; D051176:beta Catenin