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Keywords:

  • Dickkopf;
  • Wnt;
  • LRP5;
  • osteoblast;
  • osteoporosis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Wnt/β-catenin signaling has been proven to play a central role in bone biology. Unexpectedly, the Wnt antagonist Dkk2 is required for terminal osteoblast differentiation and mineralized matrix formation. We show that Dkk1, unlike Dkk2, negatively regulates osteoblast differentiation and bone formation.

Introduction: The Wnt co-receptor LRP5 is a critical regulator of bone mass. Dickkopf (Dkk) proteins act as natural Wnt antagonists by bridging LRP5/6 and Kremen, inducing the internalization of the complex. Wnt antagonists are thus expected to negatively regulation bone formation. However, Dkk2 deficiency results in increased bone, questioning the precise role of Dkks in bone metabolism.

Materials and Methods: In this study, we investigated specifically the role of Dkk1 in bone in vitro and in vivo. Using rat primary calvaria cells, we studied the effect of retroviral expression of Dkk1 on osteoblast differentiation. In addition, the effect of Dkk1 osteoblast was studied in MC3T3-E1 cells by means of recombinant protein. Finally, to address the role of Dkk1 in vivo, we analyzed the bone phenotype of Dkk1+/ animals.

Results: Retroviral expression of Dkk1 in rat primary calvaria cells resulted in a complete inhibition of osteoblast differentiation and formation of mineralized nodules, with a marked decrease in the expression of alkaline phosphatase. Dkk1 expression also increased adipocyte differentiation in these cell cultures. Recombinant murine Dkk1 (rmDkk1) inhibited spontaneous and induced osteoblast differentiation of MC3T3-E1 cells. To determine the role of Dkk1 in vivo and overcome the embryonic lethality of homozygous deletion, we studied the bone phenotype in heterozygous Dkk1-deficient mice. Structural, dynamic, and cellular analysis of bone remodeling in Dkk1+/− mice showed an increase in all bone formation parameters, with no change in bone resorption, leading to a marked increase in bone mass. Importantly, the number of osteoblasts, mineral apposition, and bone formation rate were all increased several fold.

Conclusions: We conclude that Dkk1 protein is a potent negative regulator of osteoblasts in vitro and in vivo. Given that a heterozygous decrease in Dkk1 expression is sufficient to induce a significant increase in bone mass, antagonizing Dkk1 should result in a potent anabolic effect.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Wnts are secreted cysteine-rich glycoproteins that play an essential role in embryonic development.(1) Wnts are involved in a large variety of biological processes, but their most predominant role is that of regulating cell differentiation.(2) Simultaneous binding of secreted Wnts to both frizzled (Fz) family of G protein–coupled receptors (GPCRs) and the LDL receptor–related proteins (LRP)5 and LRP6 is required for their activation of the canonical pathway.(3) In the canonical Wnt/β-catenin signaling, activation of receptors results in the inhibition of glycogen synthase kinase-3β (GSK-3β), stabilization of β-catenin, and its subsequent translocation into the nucleus where, in concert with transcription factors such as Tcf or Lef, it drives transcription of target genes.(4) The ability of several Wnts to stabilize β-catenin seems to be the basis for their proliferation and differentiation-dependent effects. To fine tune Wnt response and restrict its activity in time and space, cells also secrete several Wnt antagonists. These secreted antagonists include, on the one hand, secreted frizzled-related proteins (Sfrps) and Wnt-inhibitory factor 1 (Wif1), which act as decoy receptors antagonizing both canonical and noncanonical Wnt signaling, and on the other hand, Dickkopf (Dkk) proteins and Sclerostin/Sost, which bind LRP5/6, allowing specific inhibition of canonical Wnt signaling.(5–8)

Deregulation of the Wnt/β-catenin pathway has been implicated in several cancers and in Alzheimer's disease.(9,10) Recently, however, human genetic studies have revealed a critical role of the Wnt/β-catenin pathway in bone mass acquisition and maintenance.(11) Loss of function or gain of function mutations of the Wnt co-receptor, LRP5, lead to, respectively, decreased or increased bone mass and postnatal bone formation.(12–14) These findings have been further confirmed in both in vitro and in vivo studies. For instance, Lrp5 knockout mice totally recapitulate the bone phenotype observed in human Lrp5-mutated individuals.(15) Additional genetic evidence showing the role of Wnt signaling in bone metabolism were obtained from the identification of Sost/sclerostin as the gene responsible for sclerostosis and Van Buchem diseases.(16–19) These diseases are characterized by generalized overgrowth of bone tissue mostly visible in cranial bones and in the diaphysis of the tubular bone.

In this study, we focused our attention on Dkk1 protein, a potent secreted Wnt antagonist that specifically block Wnt/β-catenin signaling.(8) Dkk1, Dkk2, and Dkk4, but not Dkk3, are able to bind Lrp5 and 6(20–22) at the third YWTD repeat domain.(23) Contrary to all expectations, it was recently reported that Dkk2-deficient mice display an osteopenic phenotype, suggesting a more complex set of interactions in the Wnt/LRP5 pathway.(24) Increased Dkk1 expression has been also associated with lytic bone lesions in patients with multiple myeloma, suggesting that Dkk1 might inhibit osteoblast differentiation or function.(25,26) We therefore studied the impact of Dkk1 on osteoblast function and bone metabolism. Our data show that Dkk1 negatively regulates bone formation both in vitro and in vivo, and a decrease in its expression is sufficient to induce a strong anabolic response in the skeleton.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Retroviral expression constructs

The parental vectors pAP2 and pRIG-Hd have been previously described.(27) Murine Dkk1, Dkk3, osterix (Osx), and Smad1 cDNA was isolated by RT-PCR using specific oligonucleotides with appropriate restriction sites. Amplified cDNA were confirmed by sequence analysis. BamHI/BstBI, EcoRI/BglI, and XhoI/BamHI restriction enzymes were used to clone Dkk1, Dkk3, and Osx, respectively, in either pAP2 or pRIG-Hd. The dominant negative (dn)-Smad1 was obtained by directed mutagenisis of Smad1 cDNA as previously reported.(28) (dn)-Smad1 was digested with BglII/XhoI and cloned into pAP2.

Primary calvaria cell preparation and culture

Primary mouse or rat calvaria cells were obtained, respectively, from newborn calvaria mice or from 3-day-old rats. Animals were killed, and the calvaria were isolated by dissection. The calvaria were rinsed several times in PBS, and any surrounding contaminating tissue was removed. Once rinsed, the calvaria were cut into small pieces (∼1 mm in size) with a scalpel, and placed in a solution containing collagenase II (100U/ml) and trypsin (0.5%), and were digested at 37°C for 20 minutes under constant rotation. Cells obtained after the first digestion were discarded, and a second digestion under the same conditions was performed. After discarding cells obtained after the second digestion, a third and final digestion was performed under rotation with collagenase II (100 U/ml) at 37°C for 40 minutes. Cells obtained after the third digestion were centrifuged and placed in 45% DMEM, 45% α-MEM, 10% FCS, and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). Cells were expanded for 3 days before freezing. Cells at passage 2 were used for subsequent retroviral transductions, RT-QPCR analysis, or luciferase assays.

Transfections and retroviral transductions

Human embryonic kidney 293T cells were cultivated in DMEM containing 10% FCS. Cells were replated at 70% confluence in 60-mm dishes before transfection. 293T cells were transfected with 1.25 μg of pCMV-gag pol, 1.25 μg of pCMV-env, and 2.5 μg of pPRIG retroviral constructs containing the different cDNAs of interest using the classic calcium phosphate technique as previously described.(29) pEGFP-N1 (200 ng; Clontech) was added to follow the efficiency of transfection. Fresh media was added 24 h after transfection. Media containing the different nonreplicative retroviruses were obtained 48 h after transfection. These supernatants, passed through 0.22-μm Millex GP filters in the presence of 4 μM of polybrene, were used to transduce primary rat osteoblasts. A second series of transductions was repeated using media containing the different retroviruses obtained 72 h after transfection.

Transduced primary rat osteoblasts were split into one series maintained in non-differentiation media (α-MEM + 10% FCS), and a second series was placed in differentiation media (α-MEM + 10% FCS containing 10 mM β-glycerophosphate and 50 μg/ml ascorbic acid) for the times indicated in the text. Media were changed every 3 days. Once nodule formation and mineralization in control cells were observed, all cells were washed in PBS and fixed in 10% formalin in PBS for 2 h at 20°C. Fixed cells were washed extensively with distilled water and stained using the classic von Kossa method. Freshly made 5% AgNO3 was added to cells placed under light until nodules appeared black. The cells were washed at least five times in distilled water and placed in 5% sodium thiosulphate to reduce nonspecific background noise. Cells were washed in distilled water and photographed. Control cells and cells transduced with the Dkk1 retrovirus were stained in Oil red O by placing fixed cells in 0.3% Oil red O in isopropanol for 10 minutes before von Kossa coloration.

Cell proliferation measurements were performed by 5-bromodeoxy uridine (BrdU) incorporation techniques using the cell proliferation ELISA Biotrak kit (Amersham). Cell proliferation was determined according to the manufacturer's protocol.

RNA extraction and quantitative RT-PCR

Total RNA samples were obtained from calvaria cell cultures using RNAplus kit provided by Quantum. Quantitative RT- PCR (RT-QPCR) for the expression of Dkk1, Dkk2, Dkk3, ALP, Osx, Runx2, Osteocalcin (OC), Axin2, naked cuticle 2 (Nkd2), and PPARγ was performed with TaqMan PCR reagent kits in the ABI PRISM 7700 Sequence detection system (Perkin-Elmer Applied Biosystems) as described by Spinella-Jaegle et al.(30) Expression level of target genes was normalized to the level of Gapdh. All specific primers and probes were obtained from Applied Biosystems. All experiments were performed in triplicate and repeated three times. Data are presented as an average ± SD from one experiment, although results were consistent across experiments.

Dkk1 protein production and purification

Mouse Dkk1 was amplified by RT-PCR, and the nucleotide sequence was confirmed by DNA sequence analysis and subcloned as His-tagged into pcDNA3. FreeStyle 293-F cells (Invitrogen) were transfected with a His-tagged Dkk1 expression construct. Stably expressing clones were obtained after selection with blasticidine at 10 μg/ ml (Invitrogen). Subcloning was performed in 96-well plates by dilution of the cells, and Dkk1 expression was tested for all clones by dot blot analysis. Two clones were selected for high expression level (1B9 and 3F8) estimated by SDS-PAGE/Coomassie blue staining at 4–10 mg of recombinant protein per liter. Clone 1B9 was used for protein production and purification. The supernatant was collected from 1B9 clones and centrifuged at 500g for 10 minutes to discard cell debris and stored at −20°C until use. After thawing, 10× buffer was added to the CM (conditioned media) to obtain final concentrations of 50 mM Tris, pH 8.0, 100 mM NaCl, and 20 mM imidazole. Clarification of the solution was achieved by filtration on 0.22-μm GP Express PLUS membrane (Millipore). The clarified medium was loaded at 5 ml/minute (i.e., 56.6 cm/h) on a 50-ml affinity column (Chelating Sepharose Fast Flow resin; Amersham) primarily loaded with 0.5 M nickel sulfate hexahydrate solution and equilibrated in buffer A (50 mM Tris, pH 8.0, 250 mM NaCl, and 20 mM imidazole). After extensive washes in buffer A, elution of recombinant Dkk1 was conducted using imidazole gradient to 250 mM. The eluted recombinant Dkk1 was ∼90% pure as determined by SDS-PAGE stained with Coomassie blue (Fig. 3A).

Luciferase assays

COS-7 or primary calvaria cells plated in 24-well plates (DMEM, 10% FCS) at 2 × 104/cm2 were transiently transfected with the Wnt-luciferase reporter construct TOPflash (1 μg total; Upstate Biotechnology) using Fugene 6 (Life Biotechnology). Co-transfection with 20 ng of pRL-TK (Promega, Madison, WI, USA), which encodes a Renilla luciferase gene downstream of a minimal HSV-TK promoter, was systematically performed to normalize for transfection efficiency. Sixteen hours after transfection, cells were washed and cultured for 24 h in media containing 2% FBS and recombinant Wnt3a in the presence or absence of recombinant Dkk1. Controls were performed in the absence of recombinant Wnt3a. Cells were lysated, and luciferase assays were performed with the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions. Ten microliters of cell lysates was first assayed for firefly luciferase and then for Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.

MC3T3-E1 cell culture, mineralization assay, and alkaline phosphatase measurement

MC3T3-E1 clone 4 cells were kindly provided by Dr R Franceschi. Cells were cultured (5% CO2 at 37°C) in α-MEM supplemented with 10% heat-inactivated FCS. For spontaneous mineralization, MC3T3-E1 clone 4 cells were incubated in mineralization medium in 24-well culture plates at an initial density of 5 × 104 cells/cm2 in α-MEM containing 10% FCS and 50 μg/ml ascorbic acid. For mineralization in response to bone morphogenetic protein 2 (BMP-2) or sonic hedgehog (Shh), cells were cultured as indicated above except that the FCS concentration was lowered to 2% instead of 10%. BMP-2 used at 10 ng/ml and Shh used at 5 μg/ml were obtained as previously described.(30,31) For all mineralization assays, cells were cultured for 13 days, and 4 mM NaHPO4 was added 2 days before the end of the culture period to induce extracellular matrix mineralization. The mineralized matrix was stained by Alizarin red staining method as described by Stanford et al.(32) After 13 days, cells were washed with PBS and fixed in ice cold 70% ethanol for at least 1 h. Ethanol was removed, and cells were rinsed with water and stained with 40 mM Alizarin red, pH 4.2, for 10 minutes at room temperature. Stained cells were further rinsed five times with water and washed for 15 minutes in PBS with rotation to reduce nonspecific Alizarin red stain. Stained cultures were photographed, and Alizarin red fluorescence was measured on a multiplate reader (Vmax; Molecular Devices). Data are expressed as fluorescence arbitrary units.

Alkaline phosphatase (ALP) activity was determined in cell lysates using Alkaline Phosphatase Opt kit (Roche Molecular Biochemicals). Cell lysates were analyzed for protein content using microBCA Assay kit (Pierce), and ALP activity was normalized for total protein concentration.

Generation of Dkk1 mutant mice

Dkk1 heterozygotes(33) were interbred to generate Dkk1 heterozygotes (Dkk1+/−) and wildtype littermate sibling adult mice. The line was kept in a C57/Bl6 genetic background. Adults were genotyped by gene-specific PCR, using DNA from tail, with the following primers: Wt allele (416 bp), 5′-GGGAGCCTGAGTATAAAGGC-3′ and 5′-AAGAGTCTGGTACTTGTTCC-3′; mutant allele (331 bp), 5′-GAGAGGGCACAGCGATTAGGT-3′ and 5′-TACCGGTGGATGTGGAATGTG-3′.

Bone phenotyping of Dkk1+/− animals

Bone phenotype was determined in 12-week-old Dkk1+/− and wildtype littermate mice. To label bone mineralization fronts, control, Dkk1+/−, and wildtype littermate mice were given calcein (20 mg/kg; Sigma) by subcutaneous injections 10 and 3 days before death. For osteocalcin level measurements, blood was collected on the day of death. Plasma and serum samples were separated by centrifugation at 3000 rpm (750g) for 20 minutes at room temperature, and serum was stored at −20°C until analyzed. All assays were performed in duplicate. Serum osteocalcin was assayed with kits and reagents from Biomedical Technologies (Stoughton, MA, USA) as previously described.(34) The intra-assay CV was 2.0–2.3% and interassay CV was 4.0–5.0%. Urinary deoxypyridinoline cross-link levels (D-Pyr) were measured using a colorimetric assay from Pacific Biometrics (Tampa, FL, USA) and normalized to creatinine concentration (measured by Metra creatinine assay kit; QUIDEL, San Diego, CA, USA) to correct for water excretion as described by Sims et al.(34) The intra-assay CV was 3.1–4.8% and interassay CV was 4.3–8.4%.

Mouse tibias were recovered from 12-week-old mice after death and were used for tomodensitometric, histomorphometric, and densitometric analyses. For tomodensitometry, the right tibias were fixed overnight in 3.7% formaldehyde in PBS, washed in PBS, and stored in 70% ethanol. μCT scans of the metaphyseal region were performed at an isotropic resolution of 9 μm to obtain trabecular bone structural parameters. Using a 2D and 3D model and a semiautomatic contouring algorithm, we determined 3D bone volume, bone surface and trabecular thickness. 3D images were obtained on a Scanco Medical μCT scanner (μCT 20; Scanco Medical, Bassersdorf, Switzerland). A total of 450 images were obtained from each bone sample using a 512 × 512 matrix, resulting in an isotropic voxel resolution of 18 × 18 × 18 μm3. Measurements were stored in 3D image arrays with an isotropic voxel size of 9 μm. A constrained 3D Gaussian filter was used to partly suppress the noise in the volumes. The bone tissue was segmented from marrow using a global thresholding procedure. In addition to the visual assessment of structural images, morphometric indices were determined from the μCT data sets. Cortical and trabecular bone were separated using a semiautomated contour-tracking algorithm to detect the outer and inner boundaries of the cortex. In trabecular bone, basic structural metrics were measured using direct 3D morphometry.(35,36) The images were also rendered for 3D display and visualization.

For histomorphometry, the fixed samples were embedded in methylmethacrylate as previously described.(37) Four-micrometer sections were stained with von Kossa to quantify the structural parameters (i.e., bone volume, trabecular number, and thickness) and with toluidine blue to measure the cellular parameters (i.e., osteoblast and osteoclast numbers). Eight-micrometer unstained sections were used to assess the dynamic parameters (i.e., bone formation rate, mineral apposition rate). All these parameters were quantified with the Osteomeasure system (OsteoMetrics, Atlanta, GA, USA) according to standard procedures. All histomorphometric measurements were performed double blinded with respect to the treatment regimens by one individual.

The left tibia was used for densitometric analysis with the Stratec pQCT XCT Research SA+ (version 5.4B; Norland Medical Systems, White Plains, NY, USA) at 70-μm resolution. Trabecular volumetric BMD was measured by metaphyseal pQCT scans positioned 1 mm from the distal growth plate and corresponding to 6% of the total length of the tibia. The trabecular region was defined as an inner area of 60% of the total cross-sectional area.(38) Diaphyseal tibia cortical BMD (cort BMD) was measured by pQCT scans positioned at 7 mm from the distal growth plate and corresponding to 42% of the total length of the tibia. Specific mode of analysis (peel 2) was used to calculate cortical density. The default algorithm removes voxels within the trabecular part that have an attenuation coefficient below the threshold of 710 mg/cm3. The interassay CVs for the pQCT measurements were <2%.

Bone mechanical testing

The tibias were excised immediately after death and frozen at −20°C in plastic bags. During the night before mechanical testing, bones were slowly thawed at 7°C and maintained at room temperature. After the removal of the fibula, a proximal tibia compression test was performed using axial compression of the tibia plateau, the shaft being fixed in methylmethacrylate cement (Technovit 4071; Heraeus Kulzer, Wehrheim, Germany). Between the different steps of preparation, each specimen was kept immersed in a physiological solution. The mechanical resistance to failure was tested using a servo-controlled electromechanical system (Instron 1114; Instron Corp., High Wycombe, UK) with the actuator displaced at 2 mm/minute. Both displacement and load were recorded. Maximal load (N), stiffness (slope of the linear part of the curve, representing the elastic deformation, N/mm), and energy (surface under the curve, N*mm) were calculated.

Statistical analyses

All experimental data presented were obtained from three independent experiments, each in triplicate. Data were expressed as mean ± SE. Statistical differences were calculated using Student's t-test or ANOVA for multiple comparisons. p values <0.05 was considered statistically significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Overexpression of Dkk1 inhibits osteblast differentiation and enhances adipogenesis

To study the effect of Dkk1 on osteoblast differentiation, we cloned murine Dkk1 in retroviral expression vector. Osx and dominant negative Smad1 (dnSmad1) were also cloned in the same expression vector as positive and negative regulator controls of osteoblast differentiation, respectively.(28,39) Finally, Dkk3, a member of the Dickkopf family that does not inhibit Wnt signaling,(20) was also cloned in this same expression system.

Cultures of primary rat osteoblasts transduced with an empty retroviral vector and maintained in osteogenic media for 21 days resulted in the appearance of characteristic mineralized nodules (Fig. 1A). As expected, transduction of primary rat osteoblasts with Osx- or dnSmad1-expressing vectors resulted in, respectively, an increase in the number of the mineralized nodules or complete inhibition of nodule formation (Fig. 1A). We then transduced primary rat osteoblasts with retroviral constructs encoding Dkk1 and Dkk3. As shown in Fig. 1A, Dkk1 expression blocked nodule formation, whereas Dkk3 had no effect (Fig. 1A). Similar results were obtained using MC3T3-E1 cells (data not shown). RT-QPCR was performed to examine the expression of different osteoblast differentiation markers, including ALP, Runx2, OC, and Osx. Expression of all differentiation markers was strongly increased in rat primary osteoblasts cultured in osteogenic media compared with control media. As expected, retrovirally driven expression of murine Osx in primary rat osteoblasts significantly enhanced the expression level of ALP and OC and had a moderate effect on Runx2 and endogenous Osx (Fig. 1B). Transduction of osteoblasts with Dkk1 inhibited ALP expression (Fig. 1C) without affecting the expression of OC, Runx2, and Osx (Fig. 1D and data not shown). Dkk3 did not modify the expression of any of the osteoblast markers examined (Fig. 1D and data not shown).

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Figure Figure 1. Dkk1, but not Dkk3, overexpression inhibits rat primary calvaria cell mineralization, ALP, and Wnt target gene expression. (A) Primary calvaria cells isolated from 3-day-old rats were transduced at passage 2 with either control retrovirus (vector) or virus encoding for dominant negative form of Smad1 (dnSmad1), osterix (Osx), Dkk1, or Dkk3. Transduced cells were grown in the absence (CTRL) or presence of β-glycerol phosphate and ascorbic acid (β-glycP) for 21 days and then von Kossa stained. Experiments were repeated three times, and photos (magnification, ×40) are shown from one representative experiment. (A–D) Total RNA was also extracted from transduced cells, and the expression of indicated genes (ALP, Runx2, Osx, OC, or Axin2) was measured by RT-QPCR analysis and normalized to Gapdh expression. Data are presented as the relative mRNA variation in β-glycerol phosphate– and ascorbic acid–treated cells vs. control cells.

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Finally to study whether overexpression of Dkk1 or Dkk3 affects Wnt signaling in calvaria cells, we performed RT-QPCR analysis to measure Wnt/β-catenin target genes expression, Axin2 and naked cuticle 2 (Nkd2), in RNA prepared from calvaria cells transduced with control vector or with vector expression either Dkk1 or Dkk3 (as indicated above). The used genes are commonly admitted and used as a Wnt signaling reporter both in vitro and in vivo.(40,41) As shown in Fig. 1E, Axin2 expression level was significantly decreased when cells were transduced with Dkk1 expression retrovirus, but not with Dkk3. Similar data were obtained when measuring Nkd2 expression (data not shown), indicating that, in Dkk1-transduced cells, Wnt signaling activity is importantly reduced. Altogether, our data clearly indicate that Dkk1 strongly decreases osteoblast differentiation/maturation in a Runx2-independent manner.

Microscopic observation showed the appearance of adipocytic cells when the cells were transduced with Dkk1 but not Dkk3. These observations were further confirmed by Oil red O staining of lipid vacuoles (Fig. 2A). Importantly, retrovirally driven Dkk1 expression in calvaria cells resulted in a dramatic increase in PPARγ gene expression, a master gene for adipocyte differentiation(42) (Fig. 2B).

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Figure Figure 2. Overexpression of Dkk1 increases adipogenesis in rat primary calvaria cells. Primary calvaria cells isolated from 3-day-old rats were transduced with either control retrovirus (vector) or retrovirus encoding for Dkk1 or Dkk3. Transduced cells were grown in the absence or presence of β-glycerol phosphate and ascorbic acid for 21 days and analyzed for their adipocytic phenotype by either (A) red-oil stained or (B) PPARγ gene expression. For gene expression analysis, total RNA was extracted, and PPARγ mRNA was quantified by RT-QPCR, normalized on the basis of Gapdh expression, and presented as the relative mRNA expression level of cells treated with β-glycerol phosphate and ascorbic acid to control untreated cells.

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Recombinant Dkk1 protein inhibits BMP-2– and Shh-induced MC3T3-E1 differentiation

To further study the role of Dkks in osteoblast maturation, we produced and purified recombinant murine Dkk1 protein (rmDkk1; Fig. 3A). The biological activity of rmDkk1 was assessed by its capacity to inhibit Wnt signaling in COS-7 cells (Fig. 3B) and by its capacity to interact with soluble LRP5 receptor in an immunoprecipitation assay (data not shown). Under culture conditions where MC3T3-E1 cells are able to mineralize the extracellular matrix, rmDkk1 was able to block mineralization in a concentration-dependent manner as determined by Alizarin red staining (Fig. 4A). The mineralized extracellular matrix was further quantified by measuring Alizarin red fluorescence. As shown in Fig. 4B, rmDkk1 decreased fluorescence, reflecting a decrease in mineralization. This reduction was paralleled with decreased ALP activity in MC3T3-E1 cells (Fig. 4C). We further evaluated the impact of rmDkk1 on MC3T3-E1 differentiation under conditions where mineralization is enhanced by the addition of recombinant BMP-2 or Shh. As shown in Figs. 4C and 4D, Shh strongly increases MC3T3-E1 mineralization and ALP activity. Interestingly, rmDkk1 was able to block the activity of Shh on MC3T3-E1 in terms of both ALP activity and mineralization (Figs. 4C and 4D). Similar results were obtained using BMP-2 (data not shown). These results further show that Dkk1 negatively regulates osteoblast function.

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Figure Figure 3. Production and purification of recombinant murine Dkk. (A) 6His-tagged recombinant murine Dkk1 (rmDkk1) protein was purified to ∼90% purity. Forty, 20, and 10 μg of rmDkk1 were analyzed by PAGE and Coomasie blue stained. One unique band could be detected at the expected size. (B) rmDkk1 inhibits Wnt/β-catenin signaling. COS-7 cells were transiently co-transfected with TCF-1 expression construct, TOPflash, pTK-Renilla. Eighteen hours after transfection, cells were treated either with or without rWnt3a in the absence or presence of increasing concentrations of rmDkk1. Luciferase activity was determined in cell lysates after 24 h and normalized to renilla signal. All experiments were performed in triplicate.

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Figure Figure 4. rmDkk1 inhibits MC3T3-E1 cell spontaneous and Shh-induced mineralization and alkaline phosphatase activity. (A) MC3T3-E1 clone 4 cells were cultured in the mineralization medium containing 5% FCS, β-glycerol phosphate, and ascorbic acid. Cells were either left untreated (CTRL) or treated with increasing concentration of rmDkk1. Cells were cultured for 13 or 5 days to assess for mineralization and alkaline phosphatase (ALP) activity, respectively. The mineralized matrix was stained with alizarin red for calcium. Stained cells were photographed. Experiments were performed in triplicate and photos from representative wells are shown. (B) Alizarin red staining was quantified by fluorescence measurement (*p < 0.05). (C) Spontaneous ALP activity was determined in cell lysates and normalized to protein content (*p < 0.05). (C) MC3T3-E1 cells were cultured in 2% FCS in the presence of ascorbic acid and β-glycerol phosphate. Cells were either left untreated (CTRL) or treated with Shh (5 μg/ml) in the absence or presence of rmDkk1 (200 ng/ml). Cells were cultured for 13 or 5 days to assess for mineralization and ALP activity, respectively. The mineralized matrix was stained with alizarin red. Experiments were performed in triplicate, and photos from representative wells are shown. (D) ALP activity in response to Shh was determined in cell lysates and normalized to protein content (*p > 0.05).

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Mice heterozygous for Dkk1 deletion display an increased bone formation and a high bone mass phenotype

Whereas Dkk1 null animals die at birth because of a lack of most head structures, Dkk1 heterozygous mutants (Dkk1+/−) are viable.(33)Dkk1+/− mice do not differ in size or weight from their same sex wildtype littermates at birth or at death, and sexually mature female and male Dkk1+/− mice are fertile (data not shown). We have first determined the expression level of Dkk1 transcript in calvaria cells derived from both newborn Dkk1+/− and wildtype littermate animals. As shown in Fig. 5A, cells derived from Dkk1+/− animals have about 50% reduction in Dkk1 expression compared with wildtype cells. In contrast, the mRNA expression level of Dkk2 was comparable in both cell types (Fig. 5A). We determined whether reduced Dkk1 expression in calvaria cells derived from Dkk1+/− animals lead to an increase in Wnt signaling. Dkk1+/− and wildtype calvaria cells were transiently transfected with Topflash reporter construct and stimulated with either recombinant Wnt3a or a specific GSK-3β inhibitor, SB216763.(43) Our data show that, in Dkk1+/− calvaria cells, luciferase activity in response to Wnt3a was 2-fold higher than in wildtype cells, whereas SB216763, which acts downstream of the Wnt receptors, stimulates equally Wnt signaling in both cell types (Fig. 5B).

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Figure Figure 5. Cell phenotype of Dkk1+/−-derived calvaria. (A) RT-QPCR with Dkk1- and Dkk2- specific primer pairs using calvarial osteoblast mRNA harvested from Dkk1+/− and Dkk1+/+ cells. Note that Dkk1 level is reduced in Dkk1+/− compared with Dkk1+/+, whereas Dkk2 mRNA abundance is not altered in both cell types. (B) Increased responsiveness to Wnt3a in cells harvested from Dkk1+/− compared with Dkk1+/+ cells. Cells were transiently transfected with Topflash and renilla reporters and cultured in the presence of 50 ng/ml rWnt3a or 5 μM SB216763 (GSK-3β inhibitor). Luciferase activity was determined in cell lysates after 24 h and normalized to renilla signal (*significant difference from wildtype at p < 0.05). (C) Enhanced cell proliferation in Dkk1+/− cells. Dkk1+/− and Dkk1+/+ calvaria cells were cultured for the indicated number of days (d), and cell proliferation was measured by assessing BrdU incorporation. (D) Dkk1+/− and Dkk1+/+ calvaria cells were cultured in osteogenic media in the presence of BMP-2 (100 ng/ml). Dkk1+/− and Dkk1+/+ calvaria cells were cultured for the indicated cells. ALP activity was assessed in cell lysates and normalized to total protein content.

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In addition we studied whether Dkk1 deficiency affects osteoblast calvaria cells proliferation and activity. As shown in Fig. 5C, by mean of BrdU incorporation assay, at all culture times (1–7 days), Dkk1+/− cells proliferate at higher rate than wildtype littermate cells. Osteoblast differentiation of Dkk1+/− and Dkk1+/+ cells was also measured by monitoring ALP activity. When cultured in osteogenic media (i.e., ascorbic acid and β-glycerol phosphate), both cell types displayed similar ALP activity (data not shown). However, when stimulated with BMP-2, Dkk1+/− cells were two to seven times more responsive than Dkk1+/+ cells (Fig. 5D). Our data indicate that in Dkk1+/− calvaria cell expression Dkk1 is reduced by one-half, resulting in an increased responsiveness to Wnt, increased proliferation rate, and higher osteoblast activity in response to BMP-2.

We analyzed the bone phenotype in Dkk1+/− animals and compared it with wildtype littermates. Bone volume, analyzed by μCT and histomorphometry, in 12-week-old Dkk1+/− mice compared with wildtype animals was increased (Figs. 6A and 6B). This was associated with an increase in trabeculae number and thickness as well as a decrease in trabeculae spacing (Table 1). This high bone mass phenotype was observed in both males and females. Importantly, all bone formation markers were increased in Dkk1+/− mice, whereas resorption markers were comparable with wildtype. Mineralizing surface (MS) and mineral apposition rate (MAR) were both increased 2-fold in Dkk1+/−, resulting in a 4-fold increase in trabeculae bone formation rate (BFR; Fig. 6C; Table 1). The number of osteoblasts was significantly higher in Dkk1+/− compared with Dkk1+/+ (Table 1). In contrast, the number of osteoclasts as well as urinary Dpyr levels were similar in heterozygous mutant and wildtype animals (Table 1), indicating that osteoclast differentiation and bone-resorbing activity are not affected by the decreased expression of Dkk1.

Table Table 1.. Bone Structural, Dynamic, and Cellular Characteristics of Dkk1 Heterozygous Mice
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Figure Figure 6. Bone phenotype of 12-week-old Dkk1+/− mice. (A) μCT reconstruction of proximal tibias from Dkk1+/+ and Dkk1+/−, showing increased trabecular bone volume in the mutant mouse. (B) Photomicrographs of coronal sections through proximal tibias of Dkk1+/+ and Dkk1+/− mice stained with von Kossa. Note the increased numbers of trabeculae in heterozygous mutant mice. The numbers represent trabecular BV/TV of each genotype (**significant difference from wildtype at p < 0.01) (C) Fluorescent photomicrograph of bone trabecula from Dkk1+/+ and Dkk1+/− mice after calcein double labeling. Data show a significant increase in mineralization front resulting in an higher bone formation rate (BFR) in Dkk1+/− (*significant difference from wildtype at p < 0.05).

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Finally, we assessed the mechanical properties of Dkk1+/− long bones, using an axial compression test of the proximal tibia.(44)Dkk1 heterozygous mice displayed a higher maximal load and energy than wildtype littermates (Table 2). This was observed both in male and female mice. These high bone mechanical properties are associated with higher bone mass and trabecular connectivity. Taken together, these data clearly indicate that reduced Dkk1 expression leads to an increase in osteoblast differentiation and activities and enhances bone formation without any significant effect on bone resorption.

Table Table 2.. Proximal Tibia Mechanical Properties of Male and Female Dkk1+/− and Dkk1+/+ Mice
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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES

Mammalian molecular and genetic data have pointed to the critical role played by the Wnt co-receptor LRP5 in the regulation of bone formation. The fact that Dkk proteins are known to antagonize the canonical Wnt signaling by simultaneously binding to LRP5/6 and Kremen led us to examine (1) the potential impact of Dkk1 in osteoblast function by retroviral expression and the use of a recombinant Dkk1 protein and (2) the bone phenotype displayed by Dkk1 heterozygous-deficient mice. Our in vitro and in vivo data presented herein show that Dkk1 negatively affects osteoblast function.

Forced expression of Dkk1 in primary rat osteoblasts resulted in inhibition of ALP activity and nodule formation. This inhibitory activity is related to the antagonism of Wnt signaling because forced expression of Dkk3, a close homolog of Dkk1 that is unable to bind LRP5/6 and to inhibit Wnt signaling, did not affect the function of primary rat osteoblasts. Interestingly Dkk1 did not sensibly modify the expression of the osteoblast differentiation markers Runx2, OC, or Osx. This correlates with previous reports indicating that the Wnt/β-catenin pathway affects osteoblast function in a Runx2-independent manner.(15,45) An interesting finding in this study was the adipocytic commitment induced by Dkk1 in calvaria cells. This was morphologically and molecularly shown by Oil red O staining and by the increase in expression of the master adipogenic gene PPARγ. The adipogenic potential of calvaria cells has been reported,(46) and it is known that some Wnts display a high anti-adipogenic activity both in vitro and in vivo.(47–49) Our results suggest that Dkk1 inhibits the anti-adipogenic activity of Wnt proteins secreted in an autocrine manner by some calvaria cells. Given the high plasticity of immature progenitor cells and osteoblasts,(50) one can speculate that a less efficient commitment of pluripotent precursor cells into osteoblasts at the expense of adipocytes could result in the decrease or delay in the formation of mineralized nodules by calvarial cells observed in the presence of Dkk1. However, given that Dkk1 did not affect the expression of osteoblast differentiation markers such as Runx2, Osx, and OC, inhibition of mineralization is most likely to be attributed to a direct effect on the function of mature osteoblasts. In agreement with this hypothesis, purified rmDkk1 was shown to inhibit spontaneous mineralization and ALP activity of MC3T3-E1 pre-osteoblastic cells in a dose-dependent manner. Furthermore, rDkk1 also impaired ALP activity and mineralization of these cells in response to known morphogens, including BMP-2 and Shh. An increasing body of evidence supports the fact that Wnt signaling is downstream of Hedgehog- and BMP-induced osteoblast differentiation.(45,51) Indeed, previously published data from our group have showed that the capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade and that the effects of BMP2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop.(45) These findings were further supported by a comparative analysis of β-catenin conditional knockout with the Indian Hedgehog–deficient mice that places Wnt signaling downstream of Ihh signaling during early phases of osteogenesis.(51) Altogether, these in vitro data show that Dkk1 strongly inhibits osteoblast differentiation and/or function.

Our study shows that Dkk1 haploinsufficiency in mice results in a high bone mass phenotype. Reduced Dkk1 expression in Dkk1 heterozygous mice results in an increased Wnt canonical signaling. Furthermore, Dkk1+/− calvaria cells display a higher proliferation rate and increased responsiveness to BMP-2 stimulation. Our results are in concordance with genetic and in vitro evidences showing that canonical Wnt signaling promotes bone formation. The increased trabecular BMD and bone volume displayed by Dkk1+/− mice results from an increase in both the number of osteoblasts and their activity. This phenotype of high bone mass and improved trabecular connectivity observed in Dkk1+/− mice is associated with higher bone mechanical properties (maximal load and energy) of the proximal tibia. Both male and female Dkk1+/− mice display this phenotype.

Deletion of another Wnt-secreted antagonist Sfrp1 in mice also results in a preferential activation of osteoblasts, leading to enhanced trabecular bone formation.(52) Although this phenotype is similar to that found in our Dkk1+/− animals, the heightened trabecular bone mass of Sfrp1−/− mice was observed only in older animals (starting from 35 weeks old), whereas Dkk1+/− mice display a pronounced bone phenotype as early as 12 weeks of age. This difference might be attributed, at least partially, to the distinct molecular mechanisms displayed by Dkk1 and Sfrp1 in inhibiting Wnt signaling. By bridging LRP5/6 and Kremen proteins, Dkk1 only inhibits the Wnt/β-catenin canonical pathway, whereas Sfrp1 binds to a number of distinct Wnt proteins and is supposed to display a broader inhibitory effect thereby inhibiting both canonical and noncanonical pathways.(8) This raises the question of the potential contribution of the Wnt noncanonical pathway to bone metabolism. Although today there is no direct evidence showing a role of this noncanonical pathway in bone biology, this issue requires further examination.

The bone phenotype displayed by Dkk2-deficient mice has been recently reported.(24) Unexpectedly, Dkk2−/− mice are osteopenic because of major defects in mineralization. Without a significant change in the number of osteoblasts, the osteoid surface was dramatically increased and dynamic parameters of bone formation were sensibly reduced in these animals. Given that Dkk1 and Dkk2 were previously thought to have similar biological roles, the dramatic differences between the bone phenotypes displayed by Dkk1+/− and Dkk2−/− mice is intriguing. In vivo data suggest that Dkk1 and Dkk2 play opposite roles with respect to osteoblast function. Both calvarial and bone marrow–derived osteoblasts obtained from Dkk2-deficient mice showed delayed mineralization in cultures compared with wildtype osteoblasts. Although Dkk2 deficiency did not substantially affect the expression of osteocalcin and osteopontin in vivo, the expression of these two osteoblast markers was markedly reduced in cultures of calvarial osteoblasts obtained from Dkk2−/− mice in comparison with that obtained from wildtype.(24) Based on both in vivo and in vitro data, it has been suggested that Dkk2 is involved in terminal osteoblast differentiation.

A number of elements need to be taken in account to analyze the differences found in the activities of Dkk1 and Dkk2 on osteoblasts both in vitro and in vivo. First, Dkk2 has been shown to activate the Wnt/β-catenin pathway in Xenopus embryos.(53) The fact that the second cysteine-rich domain of Dkk2 has been shown to activate this cascade through LRP6(54) suggests that the agonist activity of Dkk2 in Xenopus is caused by the proteolysis of Dkk2 in this biological model. Although there is no evidence that such a proteolytic process takes place in mammals, one cannot exclude the possibility that the positive modulating activity of Dkk2 on osteoblasts both in vitro and in vivo could be partially caused by a Dkk2 fragment. Second, Dkk1 and Dkk2 are known to display different affinities for LRP5 and LRP6. Dkk1 has a higher affinity for LRP6, whereas Dkk2 interacts preferentially with LRP5.(22,55) Third and most importantly, the expression pattern of Dkk1 and Dkk2 in osteoblasts in vitro and in vivo and the regulation of the expression of these genes in response to the activation of the Wnt signaling cascade are very different. Expression of Dkk2 declines during the differentiation of calvarial osteoblasts, whereas Dkk1 gene expression is dramatically increased.(24) Furthermore the same authors showed that, in long bones, Dkk1 is primarily expressed in osteocytes, whereas Dkk2 was readily detected in osteoblasts but not osteocytes. Importantly, the expression of Dkk2, but not Dkk1, is increased in response to Wnt in osteoblasts(24) (unpublished data). It is important to mention that sclerostin/Sost, another Wnt antagonist, is also mainly expressed in osteocytes in vivo. Loss of function or downregulation of Sost leads to bone overgrowth pathologies such as sclerosteosis and Van Buchem diseases.(17–19,56) One can speculate that deletion or downregulation of Sost results in a restricted enhancement of Wnt signaling in bone tissue. In any case, the differences in the spatio-temporal expression of distinct Wnt antagonists, including Dkk1 and Dkk2, could explain, at least partially, the differences observed in vivo. Cell-specific transgenic mice or conditional knockouts of Dkk1 or Dkk2 are required to further study the impact of these proteins on different bone cell types.

The parameters of bone resorption were not sensibly modified in Dkk1+/− mice, strongly suggesting that osteoclast function is not affected in these animals. The fact that Dkk1 seems to affect mainly osteoblast biology is consistent with other previously reported transgenic models targeting distinct extracellular components of the Wnt pathway. For instance, both Lrp5 knockout and transgenic mice overexpressing either wildtype Lrp5 or high bone mass Lrp5 mutant (G171V) display only an osteoblastic phenotype.(15,57) In addition, knockout mice of a distinct Wnt antagonist, Sfrp1, also display an increase in osteoblast activity with no effect on osteoclast or resorption parameters.(52) Although osteoclast number and resorption parameters are unchanged in Dkk1+/− mice, a substantial increase in osteoclast number was found in Dkk2−/− animals. This could be caused by the delay in the terminal differentiation of Dkk2−/− osteoblasts and a subsequent increased production of osteoclast stimuli, such as RANKL and M-CSF, by these cells.(24) Selective activation of β-catenin in osteoblasts in vivo by overexpressing mutated forms of β-catenin(58) or deleting Apc(59) resulted in a dramatic reduction in the number of osteoclasts. Importantly, the control of osteoclast differentiation by Wnt signaling seems to be indirect, through osteoblasts, mainly caused by effect of this cascade on the expression of osteoprotegerin (OPG) by osteoblasts.(41,58,59)

We have shown that increasing Wnt signaling by reducing the gene expression of the Wnt antagonist Dkk1 in vivo leads to an increased bone formation and bone mass. Importantly, a 50% reduction of Dkk1 expression results in a 2-fold increase of bone volume and a 4-fold increase in BFR, indicating that a partial reduction in the activity of Dkk1 is sufficient to achieve a marked increase of osteoblast activity and bone formation while not affecting bone resorption. Interfering with Dkk1 activity and binding to LRP5/6 could therefore constitute a novel therapeutic approach to induce an anabolic response in bone.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. REFERENCES
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