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Department of Genetics and Center for Human Genetics, Case School of Medicine and University Hospitals of Cleveland, Cleveland, Ohio,1 Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, Michigan,2 Department of Medical Genetics, University and University Hospital of Antwerp, Antwerp, Belgium3
Received 7 January 2005/ Returned for modification 21 January 2005/ Accepted 16 March 2005
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For humans, two disorders affecting bone mass have been associated with mutations in LRP5. Loss-of-function mutations cause the autosomal recessive osteoporosis-pseudoglioma syndrome (OPPG) (7). Individuals with this disease have skeletal fragility, due to low bone mass, and blindness, due to impaired blood vessel regression. The second skeletal phenotype associated with LRP5 mutation is autosomal dominant high bone mass (HBM), which is caused by heterozygous missense mutations (Fig. 1) (4, 21, 32). Affected individuals in families segregating HBM can develop complications associated with excessive bone formation, such as cranial nerve compression and severe headache (34); however, they have markedly reduced risks of skeletal fracture. OPPG and HBM phenotypes have been suggested to result from altered Wnt signaling (16). Skeletal phenotypes have also been noted when other Wnt signaling components have been altered. Disruption of secreted Frizzled-related protein 1, a secreted Wnt antagonist, causes high bone mass in mice (3). Increased production of DKK1, a canonical Wnt signaling inhibitor, by myeloma cells has been shown to correlate with bone lesions in patients with multiple myeloma (30). Tissue-specific deletion of ß-catenin in skeletal precursor cells in developing mice leads to complete failure of osteoblast differentiation (12). Transgenic mice that overexpress Wnt10b in bone marrow mesenchymal progenitor cells have significantly increased bone mass, while Wnt10b null mice exhibit decreased bone mass (2). Alterations in bone mass have also been observed in mice with osteoblast-specific deletions of ß-catenin or Apc, with the former having low bone mass and the latter increased bone mass (10a). Additionally, reduced bone mass in mice having osteoblast-specific overexpression of Dkk-1 has been reported in abstract form (20a). Finally, introduction of a single Lrp6 null allele onto a Lrp5 knockout background further reduced bone mineral density in mice (9), suggesting that Lrp5 and Lrp6 have partially overlapping roles in determining bone mass.
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Here we report functional studies that utilized seven different HBM-causing missense mutations (Fig. 1), which we performed to determine how they affect Wnt signaling. We found that none of the HBM mutations were active in the absence of added Wnt. Even though different HBM mutant proteins transited the cell and reached the surface with differing efficiencies, all were able to transduce exogenously applied Wnt at levels comparable to those for WT-LRP5. However, compared to WT-LRP5, all of the HBM mutants were less inhibited by exogenously applied DKK1 protein and had reduced physical interaction with DKK1. We conclude that autocrine activation of Wnt signaling is not likely to be the general mechanism by which all missense mutations in LRP5 result in HBM phenotypes. Instead, our data support diminished regulation of the pathway by endogenous inhibitors, such as DKK1, as the most consistent mechanism for each of the HBM mutation's effects.
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MATERIALS AND METHODS |
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To generate the expression construct containing the entire extracellular domain of human LRP6 fused to the constant region of the mouse immunoglobulin G (IgG) heavy chain (LRP6N-Fc), the sequence encoding the entire human LRP6 extracellular domain was cloned into N-LRP6-mIgG (described in reference 10) to generate LRP6N-Fc. The G158V mutant of LRP6N-Fc, which is equivalent to G171V in LRP5, was generated by site-directed mutagenesis and sequence verified.
To generate a carboxy-terminal V5-tagged version of mouse Wnt1, the gene was PCR amplified from cDNA. The forward primer attached a HindIII restriction site, and the reverse primer eliminated the stop codon and inserted an EcoRI restriction site. The PCR product was cloned into the TOPO Blunt vector (Invitrogen) to generate TOPO-mWnt1. This vector was sequenced to verify the amplified product. The TOPO-mWnt1 vector was digested with HindIII and EcoRI, and the mWnt1 fragment was cloned into the pcDNA6-V5/His expression plasmid (Invitrogen) to generate mWnt1-V5.
Xi He provided a Flag-tagged human DKK1 construct (27) that we subcloned into pcDNA3.1. DKK1-V5 was made by PCR amplifying DKK1 cDNA and cloning it into the pcDNA3.1-V5/His expression vector (Invitrogen). Bernadette Holdener provided mouse MESD-C2 and RAP expression constructs (11). Ormond MacDougald provided a mouse Wnt10b construct (2), which we subcloned into pcDNA3.1. Hans Clever provided the Topflash reporter construct (20). A Renilla luciferase construct, pRL-TK, was purchased (Upstate Biotechnology).
Cell culture, expression of LRP5, LRP6, and DKK1, and collection of conditioned media. HEK293T cells and Cos7 cells (American Type Culture Collection) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Cellgro) with 10% fetal bovine serum (FBS) (Gibco-BRL). Conditioned medium (CM) containing DKK1-Flag (DKK1-CM) and conditioned medium from empty-vector-transfected cells (pcDNA3-CM) were generated by transiently transfecting 80% confluent 293T cells in six-well plates by use of Lipofectamine Plus (Invitrogen), following the manufacturer's protocol. Twenty-four hours after transfection, the culture medium was changed to 1 ml serum-free DMEM. Conditioned medium was collected 24 h later and stored frozen at –80°C until use.
LRP5N-myc recombinant protein was expressed in Cos7 or 293T cells. Cells were plated at 2 x 105 cells per well in six-well culture plates 24 h prior to transfection. Cos7 cells were transfected with 0.2 µg each of LRP5N-myc, MESD-C2, and RAP expression vectors by use of FuGENE 6 (Roche) following the manufacturer's protocol, while 293T cells were transfected by use of Lipofectamine Plus. Twenty-four hours after transfection, the culture medium was changed to serum-free DMEM. Conditioned medium was then collected 24 h later. Two percent of conditioned media and 2% of cell lysate were analyzed by immunoblot following reducing SDS-PAGE.
Control Rat2 cells and Rat2 cells stably expressing Wnt1 were provided by Anthony Brown (6). These cells were cultured in DMEM containing 10% FBS and Geneticin (50 µg/ml). Control L cells and L cells stably expressing Wnt3a were obtained from the American Type Culture Collection. These cells were also cultured in DMEM containing 10% FBS and Geneticin (50 µg/ml).
Biotinylation of cell surface protein and immunodetection of biotinylated LRP5. Cos7 cells were cultured in DMEM containing 10% FBS in 60-mm plates and, when 80% confluent, were transfected with 1.2 µg full-length LRP5-myc and 0.8 µg MESD-C2 expression vectors by use of the FuGENE 6, following the manufacturer's protocol. Forty-eight hours after transfection, cells were washed three times with ice-cold phosphate-buffered saline (PBS). Cells were incubated with PBS containing 0.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce) at 4°C for 20 min. As a negative control, transfected cells were treated with PBS lacking the biotin- cross-linker at 4°C for 20 min. The labeling reaction was stopped by three washes of chilled PBS containing 50 mM NH4Cl at 4°C. Cells were lysed in 1 ml radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate). Biotin-labeled proteins were affinity purified from 0.4 ml cell lysate mixed with 0.5 ml RIPA buffer by use of 50 µl streptavidin beads (Pierce). Samples were then resuspended in 40 µl 2x SDS-PAGE loading buffer, and 20 µl was resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions (5% ß-mercaptoethanol [BME]). Following transfer onto polyvinylidene difluoride (PVDF) membrane, LRP5-myc was immunodetected by use of a monoclonal anti-myc antibody, 9E10 (Santa Cruz Biotechnology, Inc.). As a positive control for cell surface biotinylation, ß1 integrin was immunodetected by using a monoclonal antibody MAB2252Z (Chemicon). Secondary antibody was horseradish peroxidase (HRP)-conjugated anti-mouse IgG. Immunopositive bands were visualized by chemiluminescence using the ECL-Plus Western detection system (Amersham).
Luciferase reporter assays for canonical Wnt signaling and immunodetection of LRP5 protein. Each experiment was performed in triplicate and was repeated independently on at least three separate occasions.
Assays involving 293T cells cotransfected with LRP5 and Wnt1-V5 or Wnt10b. 293T cells were plated at 2.5 x 105 cells/well in 24-well plates 24 h prior to transfection. Cells were transfected by use of Lipofectamine Plus with Topflash reporter construct (100 ng), pRL-TK (5 ng), MESD-C2 (20 ng), Wnt1-V5 (100 ng) or Wnt10b (100 ng), and full-length, untagged LRP5 (20 ng) expression vectors in 250 µl serum-free medium according to the manufacturer's protocol. As needed, empty pcDNA3.1 vector was added to make the total transfected DNA amount 250 ng. Four hours later, an additional 250 µl of DMEM containing 20% FBS was added to the cells. When DKK1-Flag was added as an exogenous inhibitor in this assay, 100 µl DKK1-CM or 100 µl pcDNA3-CM was mixed with DMEM and FBS (to make a total volume of 250 ml containing 20% FBS) and added to each well 4 h after transfection. Cells were harvested 24 h later for determination of protein expression and luciferase activity.
Assays involving 293T cells transfected with LRP5 and cultured in Wnt3a-containing conditioned medium. A transfection protocol identical to the one described above was employed, except that no Wnt1-V5 construct was included among the transfected DNAs. Serum was added back to the cells 4 h after transfection. Twenty-four hours after transfection, culture medium was replaced by 100 µl of Wnt3a-CM or control L cell-CM, along with 300 µl of DMEM containing 10% FBS. When DKK1-Flag was added as an exogenous inhibitor in this assay, 70 µl DKK1-CM or 70 µl pcDNA3-CM was mixed with Wnt3a-CM or L cell-CM, DMEM, and FBS (to make a total volume of 400 µl containing 10% FBS) and added to each well 24 h after transfection. Cells were harvested 24 h later for the determination of protein expression and luciferase activity.
Assays involving 293T cells transfected with LRP5 and cocultured with Rat2 cells stably expressing Wnt1. 293T cells were plated at 1.5 x 105 cells/well in 24-well plates 24 h prior to transfection. Cells were transfected by use of Lipofectamine Plus with Topflash reporter construct (100 ng), pRL-TK (5 ng), MESD-C2 (20 ng), and full-length, untagged LRP5 (20 ng) expression vectors in 250 µl serum-free medium according to the manufacturer's protocol. After 4 h, 250 µl of DMEM containing 20% FBS was added. Twenty hours later, the medium was removed and 400 µl DMEM with 10% FBS and either 0.4 x 105 control Rat2 cells or 0.4 x 105 Rat2 cells stably expressing Wnt1 were added to each well. When DKK1-Flag was added as an exogenous inhibitor in this assay, 180 µl DKK1-CM or 180 µl pcDNA3-CM was mixed with either 0.4 x 105 control or Wnt1-expressing Rat2 cells (to make a total volume of 400 µl containing 10% FBS) and added to each well 24 h after transfection. Cells were harvested 24 h later for determination of protein expression and luciferase activity.
Measurement of luciferase activity. Cells were lysed in 100 µl passive lysis buffer (Promega) and firefly (Topflash) luciferase activity and Renilla luciferase activity were measured using the dual luciferase assay kit (Promega) in a luminometer (Molecular Devices) following the manufacturers' instructions. Renilla luciferase activity served as an internal control for transfection efficiency.
Immunodetection of full-length, untagged LRP5. Twenty microliters of cell lysate from 100 µl of passive lysis buffer was mixed with 5 µl of 5x SDS-PAGE loading buffer. Protein was resolved by SDS-PAGE under reducing conditions (5% BME), transferred to PVDF, and immunodetected by using a monoclonal anti-LRP5/LRP6 antibody 3801-100 (BioVision). As a positive control for equal cell lysate loading, immunodetection of tubulin was performed using monoclonal antibody D-10-HRP (Santa Cruz Biotechnology, Inc.).
Coimmunoprecipitation (co-IP) of LRP5 with MESD, LRP5 with DKK1-Flag, and LRP6 with DKK1-V5. 293T cells in 60-mm culture dishes were cotransfected with 0.5 µg LRP5-myc and 0.5 µg MESD-C2 expression vectors by use of Lipofectamine Plus. As control, 0.5 µg empty pcDNA3.1 plasmid, instead of MESD-C2, was cotransfected with LRP5-myc. Cells were lysed in 1.5 ml RIPA buffer 48 h later. Lysates were freeze-thawed once before coimmunoprecipitation was performed. A total of 0.5 ml of lysate was subjected to immunoprecipitation by the addition of 10 µl of the monoclonal anti-Flag antibody M2 (Sigma) and 50 µl protein G Sepharose beads (Amersham) and rocking at 4°C for 2 h. The beads were then washed three times in high-stringency buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.5% NP-40, 0.5% Triton X-100, 0.5 mM CaCl2, 0.5 mM MgCl2) for 1 min each at 4°C and then pelleted and resuspended in 50 µl of 2x SDS-PAGE loading buffer containing 5% BME. Twenty microliters was then resolved by SDS-PAGE. Following transfer to PVDF, LRP5 protein was immunodetected by use of anti-myc monoclonal antibody 9E10, and MESD-C2 protein was immunodetected by use of anti-Flag antibody M2. Immunoprecipitation was also performed on 0.5 ml of lysate by adding 90 µl of the anti-myc antibody-conjugated agarose beads (9E10 AC) (Santa Cruz Biotechnology, Inc.) and rocking at 4°C for 2 h. Washes, sample electrophoresis, and immunodetection were performed as previously detailed.
For coimmunoprecipitation of LRP5N-myc and DKK1-Flag, conditioned medium containing LRP5N-myc was mixed with conditioned medium containing DKK1-Flag. Since HBM-LRP5 proteins were secreted into conditioned medium at various levels, immunodetection of secreted LRP5N-myc was first performed so that the amount of conditioned medium added for the co-IP experiments could be adjusted to provide equivalent amounts of secreted LRP5N-myc. This was accomplished by diluting aliquots of conditioned media containing the different LRP5N-myc proteins in serum free-media to a final volume of 1 ml. Fifty microliters of DKK1-Flag-containing conditioned medium was then added to the 1 ml of LRP5N-myc-containing medium, along with 10 µl of the goat anti-myc polyclonal antibody c-Myc(A-14)-G (Santa Cruz Biotechnology, Inc.) and 50 µl of protein G Sepharose beads (Amersham). Samples were rocked at 4°C for 5 h. Beads were then washed three times in low-stringency buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 30 s each at 4°C and then pelleted and resuspended in 50 µl of 2x SDS-PAGE loading buffer containing 5% BME. Twenty microliters was then resolved by SDS-PAGE. Following transfer to PVDF, LRP5 protein was immunodetected by using anti-myc monoclonal antibody 9E10, and DKK1-Flag protein was immunodetected by using anti-Flag antibody M2.
For coimmunoprecipitation of LRP6N-Fc and DKK1-V5, conditioned medium containing LRP6N-Fc was mixed with conditioned medium containing DKK1-V5. LRP6N-Fc- and DKK1-V5-conditioned media were made by transiently transfecting 293T cells at 80% confluence in six-well plates with 0.8 µg LRP6N-Fc and 0.2 µg MESD-C2 expression vectors or with 1 µg DKK1-V5 expression vector, respectively, using Lipofectamine Plus. Recombinant protein in 1 ml of serum-free medium was collected 48 h later. Immunodetection of secreted LRP6N-Fc was first performed so that the amount of conditioned medium added to the mixtures in the co-IP experiments could be adjusted to provide equivalent amounts of secreted LRP6N-Fc in 1 ml of serum-free DMEM. Fifty microliters of conditioned medium containing DKK1-V5 and 50 µl of anti-mouse IgG agarose beads (Sigma) were then added. Samples were incubated at 4°C for 2 h with agitation. Beads were then washed four times in low-stringency buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) for 1 min each at room temperature and then pelleted and resuspended in 50 µl of 2x SDS-PAGE loading buffer containing 5% BME. Twenty microliters was then resolved by SDS-PAGE. DKK1-V5 was immunodetected by using a horseradish peroxidase-conjugated monoclonal antibody against the V5 tag (Invitrogen); LRP6N-Fc was detected by using goat anti-mouse IgG antibody (Pierce). DKK1-V5 was used in these experiments instead of DKK1-Flag because the secondary antibody used to detect DKK1-Flag cross-reacted with an identically sized IgG fragment derived from the anti-mouse IgG agarose beads, making it difficult to determine whether DKK1-Flag had coprecipitated. This was not a problem when HRP-conjugated anti-V5 antibody was used.
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RESULTS |
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The patterns of response for the seven different HBM mutants, as assessed by induction (n-fold) of luciferase activity, are similar when Wnt10b and Wnt1 were transfected (compare Fig. 4D and A). This response is not solely due to transfection, since the pattern of response to exogenously added Wnt1 was similar to that of transfected Wnt1 (compare Fig. 4B and A). Interestingly, the pattern of HBM mutants' response to Wnt3a differed from that of Wnt1 or Wnt10b (compare Fig. 4C and A, B, and D). However, the Wnt3a pattern was the same whether the ligand was added exogenously or by transfection (data not shown). This result suggests that HBM mutations may cause subtle alterations in the affinities of the receptor for specific Wnt ligands.
All HBM-LRP5 mutants were less inhibited by DKK1 than WT-LRP5. It has previously been reported that the G171V-LRP5 mutant was less efficiently inhibited by DKK1 compared with WT-LRP5 (4). However, subsequent experiments demonstrated reduced trafficking of the G171V mutant to the cell surface and suggested that the reduced inhibition by DKK1 simply reflected there being less receptor at the cell surface; this result also suggested that the increased activity of the G171V mutant receptor was due to an intracellular interaction between the receptor and autocrine-produced Wnt ligand (37). We tested this hypothesis by adding Wnt ligand exogenously to LRP5-transfected 293T cells. All HBM mutant receptors, including G171V, were able to respond to exogenously supplied Wnt1 and Wnt3a (Fig. 4B and C), arguing against intracellular autocrine signaling as the likely mechanism. Also, as noted earlier, the patterns of responsiveness of the HBM mutants were the same whether Wnt1 was provided by cotransfection or by coculture (compare Fig. 4A and B).
Importantly, for all three Wnt ligands, we observed that each HBM mutant was less efficiently inhibited by exogenously added DKK1 in these assays than was WT-LRP5 (Fig. 4A to D). When cells were cotransfected with Wnt1-V5, the induction of luciferase activity was 70% inhibited by adding DKK1 to cells expressing WT-LRP5, whereas none of the seven HBM-LRP5 mutants was inhibited by more than 30% (Fig. 4A). Similar results were obtained when LRP5-transfected 293T cells were cocultured with Wnt1-expressing Rat2 cells. Exogenous DKK1 inhibited the induction of luciferase activity by 50% in cells expressing WT-LRP5 and by no more than 20% in any of the seven HBM mutant-expressing cells (Fig. 4B). Although the induction in luciferase activity in cells treated with Wnt3a-containing conditioned medium was smaller than that in cells treated with Wnt1-containing medium, cells expressing WT-LRP5 were more effectively inhibited by DKK1 than cells expressing any of the seven HBM mutants (Fig. 4C). The decreased inhibition by DKK1 in the HBM mutants was more clearly seen when Wnt10b was used as ligand (Fig. 4D), yielding results similar to those observed with the Wnt1 assays.
HBM-LRP5 mutants had lower affinities to DKK1 than WT-LRP5. DKK1 has previously been shown to physically interact with LRP5 and LRP6 (1, 27), leading us to hypothesize that HBM mutants have reduced interaction with DKK1. We tested this hypothesis by performing coimmunoprecipitation experiments between LRP5 and DKK1 (Fig. 5A). Human Flag-tagged DKK1 was expressed in 293T cells, and conditioned medium was mixed with conditioned medium containing secreted WT-LRP5N-myc or HBM-LRP5N-myc. For a control, we used LRP5 protein with the mutation T173M that had been identified in a patient with familial exudative vitreoretinopathy (31). Like each of the HBM mutants, this mutant is within the first EGF-like repeat domain, but it does not affect Wnt1 or Wnt10b signaling in Topflash reporter assays (our unpublished data). As shown in Fig. 5A, wild-type LRP5N-myc as well as the control T173M-LRP5N-myc comparably interacted with DKK1 protein, whereas all seven HBM-associated mutant proteins had significantly reduced interactions.
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DISCUSSION |
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The clustering of all HBM mutations within the first EGF-like repeat domain of the receptor implied that the mutations would share a common mechanism by which they affected signaling. Our data for six new HBM-LRP5 mutants agree with prior data for the G171V mutant (4) and indicate that this mechanism does not involve constitutive activation in the absence of ligand. This distinguishes HBM-LRP5 missense mutations from mutations in other receptors, such as PTHRP, which have been found to cause Jansen metaphyseal dysplasia as the result of constitutive activation (25, 26). We also show that all HBM-LRP5 mutants were able to transduce Wnt signal in the presence of added Wnt1 or Wnt3a. This is important because the Wnt interaction domain of LRP6 (and by extrapolation LRP5) is thought to involve the first and second EGF-like domains (23), and OPPG-causing missense mutations in these domains destroy the receptor's ability to transduce Wnt signal (our unpublished data). Intriguingly, we observed that several HBM-LRP5 mutants, including G171V, had greater Wnt1 and Wnt10b responsiveness than WT-LRP5 but had slightly lower Wnt3a responsiveness (Fig. 4). Consequently, it is possible that altering the affinity of the receptor to specific Wnt ligands in bone contributes to the high-bone-mass phenotype. Altered affinity of the fibroblast growth factor receptor 2 for fibroblast growth factor ligands has been implicated in causing Apert syndrome (13, 36). Other investigators did not find increased Wnt1 signal transduction by the G171V mutation compared to that of the wild type when carboxy-terminal hemagglutinin-tagged LRP5 receptors were expressed (37); however, tagging the carboxy terminus may reduce maximal signaling, since we found a carboxy-terminal myc tag impaired signaling by 50% compared to untagged receptor (our unpublished data). Therefore, the complexity of the Wnt-Frizzled-LRP signaling pathway(s) (19 different Wnt ligands, 10 different Frizzled receptors, and LRP5 and LRP6) will likely require in vivo studies using knock-in and conditional knockout alleles, rather than in vitro studies, to ultimately dissect the pathway and thereby test the validity of altered affinity to specific Wnt ligands as the mechanism for HBM-LRP5 effects.
Our results do not support the hypothesis that impaired processing of the G171V mutant and autocrine, intracellular signaling cause the HBM phenotype (37). We found that all HBM-LRP5 mutants responded similarly to Wnt1 provided either by coculture or by cotransfection (Fig. 4A and B). We also found that several HBM-LRP5s were able to transit to the cell surface (Fig. 2C) and had affinities to MESD that were similar to that of WT-LRP5 (Fig. 3). When Wnt was added to cells that had not been cotransfected with LRP5, significant canonical signaling still occurred (Fig. 4A to D), implying that endogenous LRP5 and/or LRP6 receptors transduced Wnt signal, even though they were not detectable on a Western blot (Fig. 4E). Also, the inductions (n-fold) of Wnt signaling were similar, whether 5 ng, 50 ng, or 100 ng of LRP5 expression constructs was transfected (our unpublished data). This suggests that components other than the transfected LRP5 receptors are rate limiting. Taken together, our results suggest that all transfected HBM-LRP5 mutants reached the cell surface in amounts sufficient to maximally transduce Wnt signal. Our results also demonstrate that the choice of Wnt ligand is important when interpreting in vitro studies of LRP5 signaling. Zhang et al. (37) attributed the difference in responsiveness between the G171V mutant and WT LRP5 to added Wnt3a, as opposed to cotransfected Wnt1, to there being less G171V mutant receptor reaching the cell surface. However, we found that exogenous Wnt3a was a less potent activator and exogenous Wnt1 was a more potent activator of several HBM mutants, independent of their efficiencies in reaching the cell surface (Fig. 4B and C).
We suggest that the reduced inhibition by DKK1 provides the most parsimonious explanation for the mechanism by which HBM-LRP5 mutations have their effects. Each HBM mutant was less inhibited by DKK1 than WT, whether stimulated by Wnt1 delivered by cotransfection or coculture (Fig. 4A and B), by cotransfected Wnt10b (Fig. 4D), or by Wnt3a added to the culture medium (Fig. 4C). We noticed that when Wnt3a was used, exogenous DKK1 inhibited HBM-LRP5 more efficiently than when exogenous Wnt1 was used (compare Fig. 4C and B). This result suggests the involvement of other factors at the cell surface (e.g., Kremens, Frizzled coreceptors, or as yet unidentified proteins) during inhibition of LRP5 by DKK1 when different Wnt ligands are present.
Our results demonstrate that in comparison to WT-LRP5, all mutant HBM-LRP5 proteins have reduced affinities to DKK1. When one HBM mutation, G171V, was introduced into an equivalent location in LRP6 (G158V), it also impaired the interaction between LRP6 and DKK1 (Fig. 5B). When first identified as an inhibitor of canonical Wnt signaling, DKK1 had been found to strongly interact with the third and fourth, but not the first and second, EGF-like domains of LRP6 (23). For LRP5, the first two EGF-like domains or the last two domains can bind DKK1 (37). However, Wnt signaling through LRP5 protein that lacks the third and fourth domains or just the third domain could not be inhibited by DKK1, suggesting that DKK1 binding to the third propeller domain of LRP5 is critical for regulating canonical signaling (37). While our studies have not addressed the requirement of the third domain, we clearly show that perturbation within the first domain of LRP5 or LRP6 can interfere with DKK1 binding. DKK2 has also been shown to inhibit canonical Wnt signaling by binding LRP6 (23), and two other DKK family members are present in the human genome. Our results do not exclude the possibility that the HBM phenotype results from reduced inhibition by DKKs other than DKK1 or by other as yet unidentified extracellular inhibitors of LRP5.
The existence of a naturally occurring allelic series of mutations within LRP5 that caused HBM phenotypes enabled us to mutate the specific disease-associated residues and evaluate their effects upon the Wnt signaling cascade. We found that mutations at these residues consistently interfered with inhibition of the receptor by DKK1, but not with activation of the receptor by Wnt ligand, trafficking of mutant receptor to the cell surface, or interaction of the mutant receptor with intracellular chaperones. Understanding precisely how missense mutations in the first EGF-like domain impair DKK1-mediated inhibition may lead to the rational design of pharmacologic inhibitors of this pathway that can be used to effect increases in bone mass among individuals with wild-type LRP5 receptors.
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ACKNOWLEDGMENTS |
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M. L. Warman is an investigator with the Howard Hughes Medical Institute and the recipient of a Clinical Scientist in Translational Research Award from the Burroughs Wellcome Fund. Both organizations supported this work.
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FOOTNOTES |
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30-kDa protein in this experiment. (B) Western blot of anti-Flag-immunoprecipitated protein, immunodetected by use of an anti-myc antibody (IB: myc) following reducing SDS-PAGE (4 to 15% gradient gel). Note that immunoprecipitation of MESD-Flag at high stringency comparably precipitated WT-LRP5-myc and several HBM mutants (D111Y, A214V, and A242T), while other HBM mutants were less efficiently precipitated. To ensure equal immunoprecipitation of MESD-Flag in the assay, MESD-Flag was immunodetected on the same blot by using the anti-Flag antibody (IB: flag). To ensure equal expression of LRP5-myc, cell lysates were immunodetected with anti-myc antibody (Input: LRP5-myc). Note that both assays identified the same HBM-LRP5-myc mutants as having affinities to MESD-Flag that are comparable to that of WT-LRP5-myc.
