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Keywords:

  • osteoblast;
  • Wnt;
  • β-catenin;
  • LRP5;
  • alkaline phosphatase

Abstract

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

Wnt/β-catenin signaling has recently been suggested to be involved in bone biology. The precise role of this cascade in osteoblast differentiation was examined. We show that a Wnt autocrine loop mediates the induction of alkaline phosphatase and mineralization by BMP-2 in pre-osteoblastic cells.

Introduction: Loss of function of LRP5 leads to osteoporosis (OPPG syndrome), and a specific point mutation in this same receptor results in high bone mass (HBM). Because LRP5 acts as a coreceptor for Wnt proteins, these findings suggest a crucial role for Wnt signaling in bone biology.

Materials and Methods: We have investigated the involvement of the Wnt/LRP5 cascade in osteoblast function by using the pluripotent mesenchymal cell lines C3H10T1/2, C2C12, and ST2 and the osteoblast cell line MC3T3-E1. Transfection experiments were carried out with a number of elements of the Wnt/LRP5 pathway. Measuring osteoblast and adipocyte differentiation markers addressed the effect of this cascade on osteoblast differentiation.

Results: In mesenchymal cells, only Wnt's capable of stabilizing β-catenin induced the expression of alkaline phosphatase (ALP). Wnt3a-mediated ALP induction was inhibited by overexpression of either Xdd1, dickkopf 1 (dkk1), or LRP5ΔC, indicating that canonical β-catenin signaling is responsible for this activity. The use of Noggin, a bone morphogenic protein (BMP) inhibitor, or cyclopamine, a Hedgehog inhibitor, revealed that the induction of ALP by Wnt is independent of these morphogenetic proteins and does not require de novo protein synthesis. In contrast, blocking Wnt/LRP5 signaling or protein synthesis inhibited the ability of both BMP-2 and Shh to induce ALP in mesenchymal cells. Moreover, BMP-2 enhanced Wnt1 and Wnt3a expression in our cells. In MC3T3-E1 cells, where endogenous ALP levels are maximal, antagonizing the Wnt/LRP5 pathway led to a decrease of ALP activity. In addition, overexpression of dkk1 reduced extracellular matrix mineralization in a BMP-2-dependent assay.

Conclusions: Our data strongly suggest that the capacity of BMP-2 and Shh to induce ALP relies on Wnt expression and the Wnt/LRP5 signaling cascade. Moreover the effects of BMP-2 on extracellular matrix mineralization by osteoblasts are mediated, at least in part, by the induction of a Wnt autocrine/paracrine loop. These results may help to explain the phenotype of OPPG patients and HBM.


INTRODUCTION

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

Wnt proteins constitute a family of secreted cysteine-rich glycosylated proteins involved in a large variety of modeling and remodeling processes including cell polarity, cell differentiation, and cell migration.(1,2) Secreted Wnt's bind to and activate receptor complexes consisting of the frizzled family of G-protein-coupled receptors (GPCRs) and the low-density lipoprotein (LDL) receptor-related protein (LRP)-5 and LRP-6.(3) In the canonical Wnt/β-catenin pathway, activation of receptors results in the stabilization of β-catenin and its subsequent translocation into the nucleus where, in concert with transcription factors such as Tcf or Lef, it drives the transcription of target genes.(4) The ability of several Wnt's to stabilize β-catenin seems to be the basis for their proliferation and differentiation-dependent effects. Dysfunction of the Wnt/β-catenin pathway has been implicated in several diseases such as cancer and Alzheimer's disease.(5,6) More recently, it has been shown that loss of function of the Wnt coreceptor, LRP5, in both humans and mice leads to decreased bone formation postnatally,(7–9) and a point mutation in this same receptor results in a high bone mass trait.(10,11) These reports show the crucial role that Wnt/LRP5 signal transduction plays in bone accrual during growth and the importance of this signal for the establishment of peak bone mass. However, the exact mechanisms by which Wnt signaling members regulate bone formation still remain to be elucidated.

The bone-related phenotypic abnormalities found in human or mice lacking the LRP5 protein are largely caused by an osteoblast defect.(8,9) Although the transcription factor Runx2/Cbfa1 plays a pivotal role in osteoblast differentiation,(12,13) mice deficient in LRP5 normally express Cbfa1,(8) indicating that the mechanism by which LRP5 controls osteoblast function is Runx2/Cbfa1 independent. Interestingly, although several specific osteoblast genes are controlled by Runx2/Cbfa1, alkaline phosphatase (ALP) is not. Osteoblasts are the fully differentiated skeletal cells responsible for the production of bone matrix. Osteoblasts arise from mesenchymal stem cells that are pluripotential in nature and capable of giving rise to a number of committed and restricted cell lineages including osteoblast, chondroblast, adipoblast, fibroblast, and myoblast lines. We have recently demonstrated that a number of Wnt proteins are capable of inducing the osteoblast marker ALP in mesenchymal cells.(9) The same activity has been largely described for a number of morphogenic proteins including several bone morphogenic proteins (BMPs) and hedgehog (Hh) proteins. BMP-2 increases the level of β-catenin in the nucleus of pre-osteoblastic cells and induces the expression of Wnt15, 3a, 5b, and 7,(14) suggesting the existence of an interconnection between Wnt and BMP-2 signaling and/or their ability to induce osteoblast differentiation. Here we have determined the effect of the Wnt/LRP5 cascade on osteoblast function and the role of this signaling pathway in the induction of the osteoblast phenotype by BMP-2 and Shh. Our data clearly show that Wnt/LRP5 controls the expression of ALP through the canonical β-catenin cascade and that the capacity of BMP-2 and Shh to induce the expression of ALP in mesenchymal cells is dependent on the integrity of this pathway. Moreover we have demonstrated, by using the LRP5/6-selective antagonist dkk1, that Wnt/LRP5 signaling affects mineralization of osteoblast cells.

MATERIALS AND METHODS

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

BMP-2 and N-terminal Shh production

BMP-2 was purified from Chinese hamster ovary (CHO) cells stably transfected with an expression vector encoding human BMP-2 as previously described.(15) The amino-terminal part of the murine Shh (corresponding to amino acids 25–198) was isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) and confirmed by nucleotide sequencing. The isolated sequence was subcloned into the mammalian expression vector pABWN under control of CMV enhancer/chicken β-actin promoter (pShh). pShh was transfected into mouse fibroblast L-cells (LTK-P2 cells), and stably transfected cells were established. N-Shh was purified from cells culture medium as described by Spinella-Jaegle et al.(16)

Cell culture

C3H10T1/2 and ST2 (obtained from ATCC and MC3T3-E1), and MC3T3-E1 clone 4 (kindly provided by Dr R Franceschi) cell lines were cultured (5% CO2 at 37°C) in α-MEM supplemented with 10% heat-inactivated fetal calf serum. C2C12 cells (kindly provided by Dr G Karsenty) were maintained with 5% CO2 at 37°C in Dulbecco's modified Eagle's medium supplemented with 15% fetal calf serum. For treatment or transient transfection, cells were plated at 2 × 104/cm2, and 24 h later, culture medium was changed to 2% fetal calf serum. Treatment and transfections were carried out as indicated below.

Measurement of alkaline phosphatase activity, cell proliferation, and red-oil staining

Cells were treated for indicated time with BMP-2, Shh, or BMP-2/Shh. Alkaline phosphatase (ALP) activity was determined in cell lysates using an Alkaline Phosphatase Opt kit (Roche Molecular Biochemicals). Cell lysates were analyzed for protein content using a micro-BCA Assay kit (Pierce), and ALP activity was normalized for total protein concentration.

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

Determination of adipocytic cell formation was performed by staining intracellular triglyceride droplets. C3H10T1/2 cells were cultured as indicated above and then either left unstimulated or stimulated with an IDX cocktail (insulin at 1.6 μM, dexamethasone at 0.25 μM, and 3-isobutyl-1-methylxanthine (IBMX) at 0.5 μM). The accumulation of intracellular triglyceride droplets was visualized by staining with Oil Red.

Plasmids, cell transfection, and assay for luciferase activity

Murine Dkk1, β-catenin, disheveled, Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5, Noggin, Gli3, Smad1, and LRP5 cells were isolated by RT-PCR and confirmed by nucleotide sequencing. Isolated sequences were HA-tagged subcloned into the expression plasmid pcDNA3, and expression was verified by Western blot using anti-influenza hemagglutinin (HA) antibodies. Expression of LRP5 constructs was also confirmed with an anti-LRP5/6 monoclonal antibody (BioVision). Dominant negative forms of disheveled (Xdd1) and Smad1 were generated as previously described.(16–18) Stable mutant β-catenin was generated as described by Morin et al.(19) The soluble LRP5ΔC form was generated as described by Gong et al.(9) CyclinD1 luciferase (cyclinD1/luc) reporter construct was generated as described by Leung et al.(20)

Cells were plated in 24-well plates as indicated above 24 h before transfection and transiently transfected with the indicated construct (1 μg total DNA) using DNA-lipid complex Fugene 6 (Boehringer Mannheim) according to the manufacturer's protocol. Sixteen hours after transfection, cells were washed and cultured in medium at 2% fetal calf serum for an additional 48 h under indicated conditions. Cells were assessed for ALP or luciferase activities. Controls were carried out by replacing constructs with 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. Ten microliters of cell lysate was assayed first for firefly luciferase and then for Renilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity.

RNA extraction and RT-QPCR

Cells were treated as indicated above, and total RNA was isolated from cultured cells using an isolation kit from Quantum Appligene. Quantitative RT-PCR (RT-QPCR) was performed with TaqMan PCR reagent kits in the ABI PRISM 7700 Sequence detection system (Perkin-Elmer Applied Biosystems) as described by Gallea et al.(15) Primer/probe sets for the following target genes were designed using Primer-Express V1.0 software from Perkin-Elmer: ALP, osteocalcin (OC), collagen type I, Osf2/Cbfa1, PPARγ2, Wnt1, Wnt3a, frizzled1, and frizzled9. The sequences of the primer/probe sets are shown in Table 1. All RT-QPCR reactions were performed in duplicate, and the amplification signal from the target gene was normalized to a GAPDH signal in the same reaction. Data are presented as the relative mRNA variation in treated cells versus control cells.

Table Table 1. Primer and Probe Sequences Used for TaqMan PCR Analysis
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Mineralization assay

For mineralization assays, MC3T3-E1 clone-4 cells were incubated in mineralization medium in 24-well culture plates at an initial density of 5 × 104 cells/cm2, as described by Wang et al. (α-MEM containing 10% or 2% FCS, and 50 μg/ml ascorbic acid).(21) Cells were either left untreated in the mineralization medium or were treated with BMP-2 (at indicated concentration) for 13 days. To induce extracellular matrix mineralization, 4 mM NaHPO4 was added 2 days before the end of the total 13-day culture period. The mineralized matrix was stained for calcium by Alizarin-red staining method as described by Stanford et al.(22) Briefly, after 13 days, cells were washed with PBS, followed by fixation 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 processed by five rinses with water, followed by a 15-minute wash 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.

Statistical analysis

All experimental data presented were obtained from two independent experiments, each in triplicate, and results are expressed as the mean ± SD. Comparisons between treatments were performed using Student's t-test. Values of p < 0.05 were considered statistically significant.

RESULTS

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

The canonical Wnt/β-catenin pathway induces the osteoblast marker ALP

One can distinguish between two types of Wnt proteins, β-catenin-dependent and β-catenin-independent Wnt's.(23) β-catenin-dependent Wnt's include Wnt1, Wnt2, Wnt3, and Wnt3a, whereas β-catenin-independent Wnt's include Wnt4, Wnt5a, Wnt5b, Wnt6, and Wnt7a (for more detail see http://www.stanford.edu/∼rnusse). We have tested the effect of both β-catenin-dependent and -independent Wnt's on three murine mesenchymal cell lines susceptible of acquiring the osteoblastic phenotype: C3H10T1/2, ST2, and C2C12. As depicted in Fig. 1, overexpression of Wnt3a resulted in a dramatic increase of ALP activity in C3H10T1/2, ST2, and C2C12 cells (Figs. 1A–1C). Overexpression of Wnt1 or Wnt2 resulted in an increase of ALP activity comparable with that induced by Wnt3a (data not shown). On the other hand, transient transfection of cells with the β-catenin-independent Wnt4 (data not shown) or Wnt5a (Fig. 1) did not show any significant effect on ALP expression in the cell lines used. Overexpression of β-catenin stable mutant(24) or treatment of cells with LiCl, which mimics Wnt signaling by inactivating GSK-3β thus increasing β-catenin cytoplasmic level,(25,26) significantly induced ALP expression in the three mesenchymal cell lines (data not shown).(9)

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Figure FIG. 1.. β-catenin-dependent Wnt induces alkaline phosphatase expression. (A) C3H10T1/2, (B) C2C12, and (C) ST2 cells were transiently transfected with 1 μg each of the indicated expression vector. Transfected cells were cultured for 2 days, and then ALP activity was measured in cell lysates and normalized to protein contents. Data represent the mean ± SD of representative experiment. *p < 0.05 cells transfected with Wnt3a compared with cells transfected with empty vector. **p < 0.05 cells transfected with Wnt3a/Xdd1, Wnt3a/Dkk1, or Wnt3a/LRP5ΔC compared with cells transfected with Wnt3a alone. (D) Expression used constructs in C3H10T1/2 cells. C3H10T1/2 cells were transfected with Dkk1, Xdd1, or LRP5ΔC expression construct. Untransfected cells (U) were used as control. Forty eight hours after transfection cell lysates were prepared (C), supernatants were collected. Control cells were treated likewise. Either cell lysates, supernatants, or control were separated in SDS-4 to 12% polyacrylamide gels and transferred to nitrocellulose membrane. Dkk1 and Xdd1 expression was revealed with an anti-HA antibody (left), and expression of LRP5ΔC was revealed by an anti-LRP5/6 antibody (right).

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The involvement of the canonical Wnt cascade in the induction of ALP activity was further confirmed by experiments in which transfection with a dominant-negative form of Disheveled (Xdd1), an intracellular player of the Wnt signaling,(17,18) significantly reduced the Wnt-mediated induction of ALP activity in C3H10T1/2, ST2, and C2C12 cells (Figs. 1A–1C). Furthermore, transfection with the Wnt antagonist Dkk1(27–29) markedly decreased ALP induction by Wnt3a (Figs. 1A–1C). Dkk1 has been recently demonstrated to block the interaction of Wnt with LRP5 and LRP6.(30–32) Interestingly, overexpression of a dominant negative form of LRP5 (LRP5ΔC) abolished Wnt3a-mediated ALP induction (Figs. 1A–1C). The expression of Xdd1 and the expression and secretion of Dkk1 or LRP5ΔC constructs in our cell lines was monitored by Western blot technique in both cell lysate and cell supernatant (Fig. 1D and data not shown). These results show that Wnt3a-LRP5/6 interaction is necessary for ALP expression.

We have carried out RT-QPCR in our samples to investigate the effect of Wnt3a on the expression of a series of osteoblast markers. As expected, Wnt3a induces ALP gene expression in mesenchymal cells. However, Runx2/Cbfa1, OC, and collagen type I gene expression were unaffected by Wnt3a (data not shown).

Wnt3a inhibits adipocyte markers expression in C3H10T1/2 cells

The pluripotent mesenchymal C3H10T1/2 cells can differentiate into adipocytes,(16) and Wnt proteins are known to antagonize adipocyte differentiation. We have therefore investigated whether Wnt3a overexpression affects the expression of two adipocyte markers PPARγ2 and adipocyte fatty acid binding protein (aP2) in C3H10T1/2 cells. As shown in Fig. 2A, Wnt3a clearly inhibited the expression of PPARγ2 and aP2 expression as determined by RT-QPCR analysis. Accordingly, Wnt3a completely abolished the formation of lipidic vacuoles induced by IDX, an adipogenesis stimulator, as determined by Red-Oil staining (Fig. 2B).

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Figure FIG. 2.. Wnt3a inhibits adipogenesis in C3H10T1/2 cells. (A) Wnt3a inhibits the expression of adipocyte markers aP2 and PPARγ2. C3H10T1/2 cells were either transfected with an empty vector or with the Wnt3a expressing construct. Transfected cells were then cultured for 2 days, and total RNA was extracted. The mRNA expression level of aP2 and PPARγ2 was determined by RT-QPCR. ALP mRNA was quantified and shown as a positive control of Wnt3a stimulation. The expression level of tested markers was normalized based on GAPDH expression and presented as the relative mRNA expression level of cells transfected with Wnt3a to control cells transfected with empty vector. Data represent the mean ± SD of representative experiment. *p < 0.05 cells transfected with Wnt3a compared with cells transfected with empty vector. (B) C3H10T1/2 cells were either transfected with an empty vector or with the Wnt3a expressing construct. Cells transfected with empty vector were either left untreated (i) or treated with IDX cocktail (ii), and cells transfected with Wnt3a were treated with IDX cocktail (iii). After a 5-day culture, cells were stained with oil-red O as described in the Materials and Methods section. The presented photos are representative of three distinct series of treatment.

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Wnt3a induces morphological cell transformation and enhances proliferation of C3H10T1/2 cells

When C3H10T1/2 cells were transfected with a Wnt3a expressing construct, morphological transformation was observed within 24 h (Figs. 3A and 3B). The Wnt3a overexpressing cells showed increased cell density, became more refractile, and showed elongated morphology at high magnification. In contrast, C3H10T1/2 cells transfected with control plasmid remained morphologically unchanged (Figs. 3A and 3B).

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Figure FIG. 3.. Wnt3a induces C3H10T1/2 cell transformation and enhances proliferation. C3H10T1/2 were either transfected with (A) empty vector or with a (B) Wnt3a expression construct. Transfected cells were cultured for 24 h and then photographed under light microscopy (×20). The presented photos are representative of three distinct series of transfection. (C) Wnt3a stimulated CyclinD1-luc reporter construct. C3H10T1/2 cells were transiently cotransfected with CyclinD1-luc and the empty vector or a Wnt3a expression construct. Culture medium of transfected cells was changed to 2% FCS, and culture was continued for 24 h. Luciferase activity was measured in cell lysates and was normalized to Renilla luciferase signal. (D) Wnt3a enhances cell proliferation. C3H10T1/2 cells were transiently transfected with the empty vector or with the Wnt3a expression construct. Transfected cells were cultured for the indicated time period, and cell proliferation was measured by assessing BrdU incorporation as described in the Materials and Methods section. Data represent the mean ± SD of representative experiment. *p < 0.01 Wnt3a compared with empty vector transfected cells.

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We then investigated whether Wnt3a affects C3H10T1/2 cell proliferation. We first measured the ability of Wnt3a to modulate cyclinD1 transcriptional activity, an important factor in cell cycle and proliferation, by means of cyclinD1/luc reporter construct.(20) As shown in Fig. 3C, in C3H10T1/2 control cells, the basal level of cylinD1 transcriptional activity is high and Wnt3a overexpression enhances its activity by 2.5-fold. To confirm the ability of Wnt3a to enhance C3H10T1/2 cell proliferation, we have measured cell proliferation using a BrdU incorporation assay (Fig. 3D). In a time course (1–7 days) experiment, the measurement of BrdU incorporation shows that Wnt3a does not affect C3H10T1/2 cell proliferation at early stages but enhances cell proliferation after 6 days of culture.

BMP-2 induction of ALP is dependent on the canonical Wnt pathway

The morphogens BMP-2 and Shh have been shown to induce ALP activity in C3H10T1/2 cells,(15) and it is known that the BMP, Hh, and Wnt signaling pathways are interconnected.(33–35) We therefore investigated whether the ALP-inducing activity of Wnt3a is dependent on BMP or Hh signaling. For this purpose, we assessed the effect of Noggin, a well-characterized selective BMP inhibitor,(36) and cyclopamine, which inhibits Shh signaling,(37) on Wnt3a-induced ALP expression in C3H10T1/2 cells. As expected, transfection with Noggin abolished the induction of ALP by BMP-2 (Fig. 4A), and cyclopamine dose-dependently inhibited the ALP activity induced by Shh (Fig. 4B). However, neither Noggin nor cyclopamine affected the Wnt3a-mediated ALP activity (Fig. 4). In addition, the transcriptional activity of Smad1, which is involved in the intracellular BMP signaling,(38) was not modified by Wnt3a in the presence or absence of BMP-2 (data not shown). Finally, transfection of cells with Gli3, a Shh signaling inhibitor,(39) did not affect the ALP activity mediated by Wnt3a (Fig. 4C). Altogether, these data indicate that the induction of ALP by Wnt3a is independent of BMP and Hh signaling.

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Figure FIG. 4.. Wnt3a induces ALP expression independently of BMP-2 and Shh. (A) C3H10T1/2 cells were transiently transfected with empty vector or Noggin expressing construct (pNog) and then treated with BMP-2 (100 ng/ml). Another set of cells were transiently transfected with Wnt3a and an empty vector or pNog. Control cells were only transfected with the empty vector and left untreated. Cells were cultured for 2 days, and then ALP activity was determined in cell lysates and normalized to protein content. (B) C3H10T1/2 cells were transiently transfected with empty vector or with Wnt3a construct. Transfected cells were treated with increasing concentration of cylclopamine (Cyclop) or with diluent vehicle (DMSO). Cells transfected with empty vector were additionally stimulated with Shh (10 μg/ml). Control cells were transfected with empty vector and received DMSO. Cells were cultured for 2 days, and then ALP activity was determined in cell lysates and normalized to protein content. (C) C3H10T1/2 cells were transiently transfected with empty vector or Gli3 expressing construct (pGli3) then treated with Shh (10 μg/ml). Another set of cells were transiently transfected with Wnt3a and an empty vector or pGli3. Control cells were only transfected with the empty vector and left untreated. Cells were cultured for 2 days, and then ALP activity was determined in cell lysates and normalized to protein content. Data represent the mean ± SD of representative experiment. *p < 0.001 cells treated with BMP-2 or Shh or cells transfected with Wnt3a compared with control cells. **p < 0.01 cells treated with BMP-2 or Shh in the presence of pNog, Cyclop, or pGli3 compared with BMP-2- or Shh-treated cells in the presence of empty vector and DMSO.

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We then investigated whether Wnt signaling is required for BMPs or Hh to induce ALP expression. Interestingly, overexpression of the Wnt pathway inhibitors Xdd1, dkk1, and LRP5ΔC significantly reduced, and in the case of dkk1 almost abolished, the induction of ALP activity mediated by the two morphogens (Figs. 5A and 5B). These same constructs did not affect the capacity of retinoic acid to stimulate ALP activity in C3H10T1/2 cells, indicating that the induction of ALP by retinoic nuclear receptors is independent of the Wnt pathway. Thus, our results show that the integrity of the Wnt/LRP5 pathway is necessary for Shh and BMP-2 to induce ALP in mesenchymal pluripotent cells.

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Figure FIG. 5.. BMP-2 and Shh activity independent of Wnt/β-catenin signaling. (A) C3H10T1/2 cells were transiently transfected with empty vector, Xdd1 expressing vector, Dkk1 expressing vector, or LRP5ΔC expressing vector. Transfected cells were either left unstimulated or (A) stimulated with BMP-2 (100 ng/ml), (B) Shh (10 μg/ml), or (C) retinoic acid (RA at 0.1 μM). Cells were cultured for 2 days, and then ALP activity was determined in cell lysates and normalized to protein content. Data represent the mean ± SD of representative experiment (p < 0.05). *p < 0.001 cells with empty vector and treated with BMP-2, Shh, or RA or cells transfected with Wnt3a compared with untreated transfected cells. **p < 0.01 cells transfected with expression construct and treated with BMP-2 or Shh compared with BMP-2 or Shh treated cells transfected with empty vector.

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BMP-2 induces the expression of Wnt's

One possible explanation for the results described above is that Wnt-mediated signaling might directly control the expression of ALP. Pretreatment of C3H10T1/2 or C2C12 with the inhibitor of protein synthesis cycloheximide did not prevent the induction of ALP mRNA expression mediated by LiCl treatment (data not shown) and Wnt3a (Fig. 6). In contrast, the induction of ALP gene expression by BMP-2 was inhibited by cycloheximide (Fig. 6), indicating that de novo synthesis is necessary for this morphogen to induce ALP gene expression.

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Figure FIG. 6.. BMP-2, but not Wnt/β-catenin, signaling requires de novo protein synthesis to induce ALP expression. C3H10T1/2 cells were incubated for 1 h with cycloheximide (CHX, 1 μg/ml) or with solvent vehicle (DMSO) and then either left unstimulated or stimulated with BMP-2 (100 ng/ml) or Wnt3a (transfected with 1 μg of Wnt3a expression construct). Six hours after stimulation, total RNA was extracted, and the mRNA expression level of ALP was determined by RT-QPCR, normalized based on GAPDH expression, and presented as the relative mRNA expression level of treated cells to untreated control cells. Data represent the mean ± SD (p < 0.05) of representative experiment.

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We then examined whether BMP-2 is capable of inducing the expression of Wnt's in C3H10T1/2 and ST2 cells. As shown in Fig. 7A, Wnt3a gene expression was increased by BMP-2 in both C3H10T1/2 and ST2 cells. Wnt1 gene expression was also found to be upregulated, but only in C3H10T1/2. In addition, we also investigated the expression of Wnt receptors, frizzleds (1–9), and LRP5 in response to BMP-2. LRP5 was expressed in the absence of BMP-2 and was not affected by this stimulus (data not shown). Our data show that only frizzled-1 (Fz1) and frizzled-9 (Fz9) expression was increased in the presence of BMP-2 (Fig. 7B). Altogether, our results strongly suggest that the effect of BMP-2 on ALP expression is mediated by a Wnt autocrine/paracrine loop.

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Figure FIG. 7.. BMP-2 induces Wnt and Frizzled expression. C3H10T1/2 and ST2 cells were stimulated with BMP-2 (100 ng/ml) for 24 h, and total RNA was then extracted. Total RNA was also prepared from unstimulated control cells. The mRNA expression level of Wnt1, Wnt3a, Frizzled-1 (Fz1), and Frizzled-9 (Fz9) were determined by RT-QPCR, normalized based on GAPDH expression, and presented as the relative mRNA expression level of treated cells to untreated control cells. Data represent the mean ± SD of representative experiment (*p < 0.05).

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ALP expression and mineralization of osteoblast-like cells MC3T3-E1 is controlled by Wnt signaling

To further investigate the role of Wnt/β-catenin signaling in osteoblast maturation, we examined the effects of Wnt3a in the osteoblastic-like cell line MC3T3-E1. As shown in Fig. 8A, Wnt3a overexpression did not modify the ALP activity already displayed by MC3T3-E1 cells. Overexpression of other Wnt proteins, such as Wnt1 and Wnt2, had no effect on ALP expression in these cells (data not shown). Although Wnt proteins had no effect on the already maximal ALP levels in MC3T3-E1 cells, Xdd1, LRP5ΔC, and Dkk1 significantly reduced the endogenous ALP activity displayed by these cells (Fig. 8A). We have therefore assessed the expression level of several Wnt mRNA in MC3T3-E1 cells. MC3T3-E1 cells were cultured for different time periods (2–5 days), total RNA was extracted, and the expression level of Wnt1, Wnt2, and Wnt3a was determined by RT-QPCR. The 2-day time-point was used as the reference point. ALP expression was found to be strongly and continuously increased over the time course (data not shown), as expected, with a cell committed toward the osteoblast phenotype. As shown in Fig. 8B, Wnt3a, but not Wnt1 or Wnt2, expression significantly increases over the MC3T3-E1 time course. This strongly supports the hypothesis that a Wnt/β-catenin autocrine/paracrine loop controls the expression of ALP in osteoblastic cells.

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Figure FIG. 8.. An autocrine/paracrine Wnt loop controls alkaline expression in the mature osteoblast. (A) MC3T3-E1 cells were transiently transfected with empty vector or one of the following expression constructs, Wnt3a, Xdd1, Dkk1, or LRP5ΔC. Transfected cells were cultured for 5 days, and then ALP activity was determined in cell lysates and normalized to protein content. Data represent the mean ± SD of representative experiment (*p < 0.05). (B) MC3T3-E1 cells were cultured for 2, 3, 4, and 5 days, and then total RNA was extracted. The mRNA expression level of Wnt3a was determined by RT-QPCR, normalized based on GAPDH expression, and presented as the relative mRNA expression level of 3-, 4-, or 5-day culture cells compared with 2-day culture cells (considered as reference point). Data represent the mean ± SD of representative experiment (*p < 0.05). (C) MC3T3-E1 clone 4 cells were cultured in the mineralization medium containing 2% FCS and ascorbic acid for 13 days. Cells were either left untreated (CTRL) or treated with BMP-2 at indicated concentration. 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. (D) Mineralization assay on MC3T3-E1 cells were transfected with empty vector (1 μg) or with Dkk1 expression construct (0.5 and 1 μg). After transfection, a mineralization assay was performed in the presence of BMP-2 (10 ng/ml) as described above, and alizarin red staining was quantified by fluorescence measurement as described in the Materials and Methods section. Data represent the mean ± SD of representative experiment (*p < 0.05).

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Although Wnt3a did not sensibly modify the mineralization capacity of MC3T3-E1 cells, we decided to evaluate the impact of inhibiting this signaling cascade on the mineralization of displayed by these cells in experimental conditions in which mineralization is dependent on exogenous BMP-2.(40) As shown in Fig. 8C, whereas no mineralization could be observed after 13 days of culture in the absence of BMP-2, 2.5–10 ng of BMP-2 clearly induced mineral nodules in these cells. Mineralization induced by BMP-2 could be quantified by mean Alizarin-red labeling and fluorescence measurement (Fig. 8D). Interestingly, transient overexpression of dkk1 in these cells significantly reduced the mineralizing capacity (Fig. 8D). Our results indicate that the Wnt/β-catenin pathway regulates osteoblast mineralization.

DISCUSSION

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

The recent discovery of the role of the Wnt coreceptor LRP5 in bone formation strongly suggests an involvement of the Wnt signaling pathway in skeletal biology.(8–11) Although different models show that Wnt signaling through the LRP5 receptor mainly affects osteoblast function, the precise molecular mechanisms remain to be elucidated. To gain further understanding of the control of osteoblast function by Wnt signaling, we investigated the effect of Wnt in both osteoblast commitment and differentiation by overexpressing different Wnt proteins in mesenchymal pluripotent cell lines capable of differentiating into the osteoblast lineage. Results presented herein clearly show that β-catenin-dependent Wnt's display the ability of inducing ALP expression in mesenchymal cells. The fact that LiCl was able to mimic Wnt ability to induce ALP expression and that dominant-negative disheveled inhibited Wnt ability to induce ALP expression clearly show that the capacity of Wnt's to induce ALP relies on the canonical β-catenin cascade. Moreover, the inhibition of Wnt effects, with either the LRP5/6 inhibitor dkk1 or a LRP5 dominant negative, highlight the crucial role that LRP5 plays in the induction of ALP expression by Wnt's.

Very interestingly, impairing Wnt/β-catenin signaling resulted in a significant reduction in the capacity of BMP-2 and Shh to induce ALP in these cells. These data strongly suggest that the Wnt/β-catenin pathway is required for the morphogenetic-mediated induction of ALP in mesenchymal cells. Further support for the requirement of Wnt signaling for the activity of BMP-2 activity comes from data showing that de novo protein synthesis is necessary for BMP-2 to induce ALP gene expression and that BMP-2 induces the expression of Wnt1 and Wnt3a in C3H10T1/2 cells. Our observation is consistent with the recent report that BMP-2 treatment upregulates expression of Wnt3a in high-density cultures of C3H10T1/2 cells.(41) Moreover, Hh signaling has also been shown to control the expression of Wnt proteins: Wnt5a is a target of Shh in hair follicle morphogenesis(42) and Gli1 (a crucial player in the mediation of Hedgehog signaling) has been shown to consistently induce a distinct set of Wnt genes in animal cap explants and skin tumors.(43) Very interestingly, microarray analysis of gene expression profile in different cell types demonstrated that Wnt proteins induce several genes that have been reported previously as BMP-2 target genes.(44,45) From these findings, it is expected that some activities of BMP-2 or Hh can be attributed to the effects of these proteins on Wnt gene expression. Our data suggest that BMP-2 and Shh do not directly regulate ALP expression but do so indirectly through the Wnt signal cascade. Figure 9 depicts a conceptual model for the proposed Wnt autocrine loop controlling ALP expression.

thumbnail image

Figure FIG. 9.. Conceptual figure model for a Wnt autocrine loop.

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Although Wnt overexpression did not affect the already high endogenous ALP activity displayed by the osteoblastic cells MC3T3-E1, RT-QPCR clearly shows that these cells endogenously express Wnt3a. In addition, disruption of Wnt signaling by overexpressing Dkk1 significantly reduced both ALP activity levels and the capacity of mineralization of these cells, showing the existence of an autocrine/paracrine loop in osteoblasts. Consistently, in vitro mineralization of the extracellular matrix by LRP5−/− mouse-derived osteoblasts was delayed compared with osteoblasts from wild-type animals.(8) This is due either to a functional defect in osteoblasts deprived from LRP5 or an abnormal proliferation rate in knockout animal-derived cells.(8) Although Wnt3a significantly enhances mesenchymal cell proliferation, this effect occurs very late compared with BMP-2. Our data concerning the control of ALP by Wnt signaling suggest that the absence of LRP5 affects the osteoblast function by reducing ALP activity, and subsequently, the mineralization capacity of these cells. It is important to point out that it has been recently reported that transgenic mice expressing the human high bone mass gene display an enhanced ALP staining in osteoblasts.(46) Therefore, the control of ALP expression by Wnt signaling in both pluripotent mesenchymal cells and osteoblasts might partially explain the skeletal phenotypes occurring in human or mice displaying LRP5 mutations.

In contrast with ALP, other osteoblast markers such as Runx2/Cbfa1 and osteocalcin were not affected by Wnt overexpression. This is consistent with the normal expression of these two osteoblast markers reported in LRP5−/− mice(8) and strongly suggests that Wnt signaling controls osteoblast function in a Runx2/Cbfa1-independent manner. Although elevated levels of serum osteocalcin have been found in a kindred affected with high bone mass disorder,(11) no osteocalcin data have been reported from transgenic mice overexpressing the human LRP5 HBM mutation.(46) It would be interesting to measure osteocalcin levels in these transgenic mice but also in other human kindred displaying either the HBM trait or OPPG phenotype. In any case, our results suggest that Wnt signaling regulates osteoblast commitment and function without interfering with Runx2/Cbfa1 or osteocalcin expression, and it constitutes a Runx2/Cbfa1-independent pathway crucial for osteoblast maturation. It has been previously reported that Runx2 overexpression induces ALP gene expression in C3H10T1/2 cells.(47) In the case of C2C12 cells, Lee et al.(48) found a slight increase in ALP activity in cells overexpressing Runx2, whereas Zhang et al.(49) did not observe detectable levels of ALP activity in C2C12 stably expressing Runx2. In our hands, Runx2 overexpression did not induce any detectable ALP activity in these two cell lines. One possibility is that cell lines used in other studies could spontaneously secrete some BMP proteins. In this case, Runx2 overexpression can enhance ALP activity. Actually a cooperation between Smads and Runx2 has been suggested by Zhang et al.(49) In any case, our experimental data using dkk1 clearly show that the integrity of Wnt/LRP signaling cascade is necessary for induction of ALP by BMP-2.

We also present evidence here that Wnt3a can compromise the adipocytic commitment of the pluripotent mesenchymal cell line C3H10T1/2. Importantly, Wnt3a dramatically reduced the levels of PPARγ2 and aP2, two genes playing a central role in adipogenesis. Although it is well established that Wnt signaling inhibits adipogenesis,(50) this is the first report to our knowledge showing the inhibition of adipogenesis in cells displaying an osteoblastic potential. Whereas BMP-2 has been shown to induce differentiation of C3H10T1/2 cells into adipocyte and osteoblasts,(21) Hh(16) and Wnt3a (this study) display opposite actions in adipogenesis and osteogenesis. Our hypothesis is that signaling cascades triggered by these proteins lead, respectively, to the repression of adipocyte master genes and activation of genes playing crucial roles in osteoblast differentiation such as ALP. Osteoblasts and adipocytes differentiate from the same progenitor cells, and pathways that regulate osteoblastic differentiation versus adipogenesis are of high importance. Total marrow fat increases with age, and there is an inverse relationship between marrow adipocytes and osteoblasts with aging.(51,52) The number of mesenchymal stem cells with osteogenic potential decreases early during aging in humans and may be responsible for the age-related reduction in osteoblast number.(53) Importantly, it has been demonstrated that cells cultured from human trabecular bone are not only osteogenic but are also able to undergo adipocytic differentiation under defined culture conditions.(54) It is therefore attractive to speculate that Wnt signaling constitutes one of the mechanisms to control the relative commitment of cells into either the osteoblast or the adipocytic lineage. Further investigation is required to determine the possibility of a switch from adipocyte to osteoblast commitment in a given cell. Interestingly, in the ΔFosB transgenic model (increased bone formation and inhibited adipogenesis), osteosclerosis has been shown to be independent of decreased adipogenesis.(55)

Few anabolic agents have been developed as therapeutics for skeletal disorders. Because of the crucial role of the Wnt signaling pathway for osteoblast activity, this cascade might constitute an important new target for anabolic drug development. To discover or design drugs selectively affecting this pathway, we need to elucidate the mechanisms by which the Wnt cascade affects pre-osteoblasts and osteoblast function. In this study, we contribute to the understanding of these molecular effects by showing (1) the crucial role that Wnt signaling plays in both the acquisition of ALP gene expression by mesenchymal pluripotent cell lines and the control of ALP expression and mineralization of osteoblasts, (2) the importance of LRP5/Wnt signaling in the morphogenetic protein-mediated induction of ALP in mesenchymal cells, with the consecutive impact in the commitment of these pluripotent cells into the osteoblastic lineage, and (3) the effect of Wnt cascade in inhibiting adipogenesis in mesenchymal pluripotent cells.

Acknowledgements

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

We would like to thank Dr Chantale Bardelay and M Berbard Doucet for help in adipocytic cell staining. We are also indebted to Veronique Stiot for technical help.

REFERENCES

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  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. REFERENCES
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