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.

FIGURE 1.

LRP4 is a direct SOST interaction partner. A, LRP4 was identified as sclerostin interaction partner by tandem affinity purification. C-terminal TAP-tagged sclerostin was expressed into HEK293T and affinity-purified from a cellular membrane fraction. The purified material was then loaded on an SDS gel and stained with Coomassie Blue as exemplified for one gel lane. The interaction partners were identified through mass spectrometry. B, sclerostin interacts with LRP4 in an ELISA. Sclerostin was precoated prior to the addition of recombinant LRP4 ECD. After binding and washing, LRP4 was detected by anti-Fc antibody coupled to alkaline phosphatase (AP). The left panel shows an LRP4 dose-response curve with a fixed sclerostin dose of 200 nm. The right panel shows a sclerostin dose-response curve with a fixed LRP4 dose of 20 nm. Interaction is shown as alkaline phosphatase activity (arbitrary units). C, sclerostin antibody disrupts a preformed sclerostin LRP4 complex. Binding of sclerostin (200 nm) to LRP4 (5 nm) was challenged with control antibody or antibody against sclerostin in a dose-response manner. Detection was as described in B. D, sclerostin interacts with LRP4 in surface plasmon resonance. Binding of sclerostin (four concentrations, 6.25, 12.5, 25, and 50 nm, are shown in gray) toward immobilized LRP4 was measured. Sensograms (shown in black) were fitted using a 1:1 binding model. RU, response units. Error bars, S.E.

FIGURE 2.

LRP4 is a specific facilitator of sclerostin-mediated inhibition of Wnt1/β-catenin signaling. A, LRP4 expression specifically facilitates sclerostin inhibitory action on Wnt1/β-catenin signaling. HEK293 cells were transiently transfected with LRP4, STF-LUC reporter plasmid, and Wnt signaling-inducing (Wnt1 + LRP5) plasmids. Five h after transfection, sclerostin or DKK1 was added in a dose-dependent manner for an additional 19 h. Cells were resuspended in lysis buffer, and luciferase levels were measured. B, down-regulation of LRP4 diminishes sclerostin action on Wnt1/β-catenin signaling. Stable HEK293-Wnt1-STF cells were transfected with control siRNA or siRNA against LRP4 for 48 h prior to a 24-h incubation with sclerostin or DKK1. *, p < 0.05 versus SOST in control. C, LRP4 extracellular domain is required to mediate the sclerostin facilitator effect. Membrane-anchored LRP4 truncation mutants (LRP4Δcytoplasm and LRP4ΔECD) were compared with full-length LRP4, as described in A. Error bars, S.E.

FIGURE 3.

LRP4 is expressed in human bone, and Lrp4 silencing blunts sclerostin-mediated inhibition of in vitro bone mineralization. A, LRP4 protein is expressed in human osteoblasts (arrowheads) and osteocytes (arrows). Immunohistochemistry of LRP4 in human femoral neck from a male subject aged 75 is shown. Scale bar, 50 μm. B, LRP4 RNA is expressed in samples from human femoral neck. RNA was extracted from a female subject aged 65 and a male subject aged 80. PTH1R, LRP4, SOST, LRP5, and LRP6 RNA levels were assessed by real-time quantitative PCR and normalized with GAPDH, and relative expression compared with PTH1R is shown. C, knockdown of LRP4 in Kusa-A1 blocks the inhibitory effect of sclerostin on in vitro mineralization activity. Kusa-A1 cells were transduced with lentiviral particles harboring shRNA against Lrp4. Reduction of Lrp4 RNA levels was assessed by real-time quantitative PCR, prior to the addition of an osteoblastic differentiation medium. Calcium content was assessed 4 days later. Error bars, S.E.

FIGURE 4.

Clinical features and LRP4 mutation analysis of a Greek female (A–C) and a Spanish male sclerosteosis patient (D–G). A, front view of the patient's face showing right facial asymmetry. B, dysplastic finger with shortening and nail malformation with unguis bipartitus. C, partial sequence chromatogram displaying the DNA sequence of the patient. The arrowhead indicates the presence of a homozygous c.3508C→T missense mutation (in exon 26 of the LRP4 gene), resulting in a R1170W substitution. D, plain radiographs of the hands; modeling defect of the metacarpal bones and complex fusion anomaly of the individual bones of the hands. E, plain radiograph of the skull (lateral view). Shown is extensive sclerosis of the calvaria, maxilla, mandible, and cervical spine. F, plain radiograph of the left lower leg. Shown is thickening of the cortical bone of the diaphysis of the tibia with extension into the proximal metaphysis. G, left, family pedigree. All of the proband's relatives are unaffected. Direct sequencing analysis of the proband demonstrated a heterozygous c.3557G→C missense mutation (in exon 27), resulting in a W1186S substitution (arrowhead). The mutation was absent in the DNA of both the parents, suggesting that the mutation is a de novo mutation. The mutation is not segregated to either child. N.D., mutation not detected. Right, partial sequence chromatogram displaying the cDNA (mRNA) sequence of the Spanish male, confirming the heterozygous presence of the c.3557G→C missense mutation. The numbering refers to the transcript with ID ENST00000378623 in the Ensembl data base.

FIGURE 5.

LRP4 mutations (R1170W and W1186S) impair sclerostin interaction and concomitant sclerostin-enhancer function. A, expression levels of LRP4 mutants (R1170W and W1186S) are similar to wild type LRP4. HEK293 were transfected with LRP4 wild type and mutant expression vectors and subjected to membrane-specific protein extracts (top; quantification compared with WT 100%: R1170W 95% and W1186S 104%) or cell surface proteins isolated by biotinylation (bottom; quantification compared with WT 100%: R1170W 113% and W1186S 114%) prior to anti-LRP4 immunoblotting. B, LRP4 mutants (R1170W and W1186S) have impaired sclerostin enhancer function in Wnt1/β-catenin signaling inhibition. HEK293 cells were transiently transfected with LRP4 wild type and mutants, STF-LUC reporter plasmid, and Wnt signaling-inducing (Wnt1 with or without LRP5) plasmids. Five h after transfection, sclerostin was added in a dose-dependent manner for an additional 19 h. Cells were resuspended in lysis buffer, and luciferase levels were measured. C, mutations (R1170W and W1186S) in LRP4 lead to reduced sclerostin binding properties. Conditioned medium from HEK293 cells transfected for 4 days with wild type and mutant LRP4 ECD were harvested. Levels of secreted proteins were assessed by immunoblotting, and adjusted amounts were used as a source of LRP4 in the sclerostin LRP4 interaction ELISA, as shown in Fig. 1B. *, p < 0.05; **, p < 0.01 versus LRP4 WT. D, co-localization of LRP4 mutations (R1170W and W1186S) at the surface of propeller 3 based on the structural model. E, dominant negative effect of LRP4 mutants (R1170W and W1186S) on the SOST LRP4 wild type interaction. A combination of mutant LRP4 and wild type LRP4 was used as a source of LRP4 in the sclerostin LRP4 ELISA. **, p < 0.01 versus LRP4 WT plus medium. Error bars, S.E.