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Endocrinology, doi:10.1210/en.2007-0520
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Endocrinology Vol. 148, No. 11 5323-5330
Copyright © 2007 by The Endocrine Society

Essential Role of Wnt3a-Mediated Activation of Mitogen-Activated Protein Kinase p38 for the Stimulation of Alkaline Phosphatase Activity and Matrix Mineralization in C3H10T1/2 Mesenchymal Cells

Joseph Caverzasio and Danielle Manen

Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, CH-1211 Geneva 14, Switzerland

Address all correspondence and requests for reprints to: Prof. Joseph Caverzasio, Service of Bone Diseases, Department of Rehabilitation and Geriatrics, University Hospital of Geneva, CH-1211 Geneva 14, Switzerland. E-mail: Joseph.Caverzasio{at}medecine.unige.ch.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Signaling pathways involved in the development of osteoprogenitors induced by Wnts remain poorly understood. In this study, we investigated the role of MAPKs in the development of mesenchymal cells into osteoprogenitors. In C3H10T1/2 mesenchymal cells, Wnt3a induced a rapid and transient activation of MAPKs p38 and ERK. Dickkopf 1, a selective antagonist of Wnt proteins binding to low-density lipoprotein-receptor-related protein-5/6 did not influence activation of p38 and ERK induced by Wnt3a. A MAPK kinase-1/2 (MEK1/2) inhibitor blocked, whereas a p38 inhibitor had no effect on, Wnt3a-induced cell proliferation. In contrast, both inhibitors significantly reduced alkaline phosphatase stimulation with a more pronounced effect of the p38 inhibitor. The p38 inhibitor also blunted nodule mineralization induced by Wnt3a. Associated with these effects, β-catenin transcriptional activity, assessed with the TOPflash system, was dose-dependently decreased by the p38 but not by the ERK inhibitor. Both the reduced alkaline phosphatase stimulation and blunting of β-catenin transcriptional activity were mimicked by expression of dominant-negative (dn) p38 and dnMEK 3/6. Inhibition of β-catenin transcriptional activity by the p38 inhibitor as well as by dnp38 and dnMEK 3/6 molecules were not associated with changes in cytosolic and nuclear β-catenin levels induced by Wnt3a. In conclusion, Wnt3a activates ERK and p38 in mesenchymal C3H10T1/2 cells by a low-density lipoprotein-receptor-related protein-5/6-independent mechanism. Activation of p38 regulates alkaline phosphatase activity and nodule mineralization induced by Wnt3a probably by interacting with β-catenin transcriptional activity. These observations suggest that MAPKs ERK and p38 are probably essential pathways activated by Wnt proteins for the development of mesenchymal cells into osteoprogenitors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WNTS ARE SECRETED lipid-modified signaling proteins that activate cell surface receptors and regulate a variety of cellular activities including cell fate determination, proliferation, migration, polarity, and gene expression (1, 2).

Genetic studies in humans and mice have determined that low-density lipoprotein-receptor-related protein-5 (LRP5)/Wnt signaling plays a major role in the control of bone mass. Mutations in LRP5 lead to disorders associated with either low (3, 4, 5) or high bone mass (6, 7, 8, 9). LRP5 mutations affect bone density by altering osteoblast number and bone accrual with levels of bone turnover and metabolic markers being normal in osteoporosis pseudoglioma and high bone mass patients (3, 4, 6). Lrp5–/– animals have fewer osteoblasts and a 50% reduction in bone formation and matrix apposition rates as well as a decrease in colony forming unit-fibroblast bone marrow progenitor cells producing alkaline phosphatase (ALP) indicating that Wnts proteins stimulates the proliferation and the differentiation of osteoprogenitors (4). Complementary information was obtained in transgenic mice expressing a gain-of-function mutation Lrp5G171V in which more functioning and fewer apoptotic osteoblasts, increased mineralizing surfaces, and higher ALP levels were reported (9). Recent studies demonstrated that Wnt/β-catenin signaling represents a major mechanism for the differentiation of mesenchymal cell precursors into osteoblasts (10, 11, 12, 13). Conditional deletion of β-catenin, the central molecule of Wnt signaling, in limb and head mesenchyme during early embryonic development was shown to result in arrest of osteoblastic differentiation and lack of mature osteoblasts in membranous bones (12, 13). In the absence of β-catenin, osteochondroprogenitors differentiated into chondrocytes instead of osteoblasts (13). Experiments with loss- and gain-of-function mutation alleles of β-catenin also demonstrated that β-catenin is required at an early stage to repress chondrocytic differentiation (13). All these data indicate that Wnt proteins are critical regulators of skeletal development. The molecular mechanisms by which Wnt proteins stimulate the proliferation and the differentiation of mesenchymal precursors into osteoblastic cells remain poorly understood and is of potential interest for the discovery of new targets for the treatment of bone diseases.

Recent studies indicate that canonical Wnts can directly activate the MAPK ERK (14, 15). In fibroblasts, Wnt3a activates the ERK pathway via Ras, Raf1, MAPK kinase (MEK) and plays an important role in Wnt-induced cell proliferation (14). In uncommitted osteoblast progenitors and differentiated osteoblasts, Wnt-3a prevents apoptosis by β-catenin-dependent and β-catenin-independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase (PI3K)/AKT (15). Thus, in addition to the β-catenin pathway, canonical Wnts also activate the MAPK ERK for inducing cell proliferation. Whether they also trigger activation of other MAPKs for the regulation of osteoprogenitors is unknown. In the present study, we found that, in addition to the canonical β-catenin and the ERK pathways, Wnt3a also activates p38 in C3H10T1/2 cells. Functional analyses indicate that activation of p38 interacts with the β-catenin pathway and influences the development of C3H10T1/2 cells into skeletogenic cells. We also found that activation of p38 is independent of LRP5/6. Whether activation of this pathway by Wnt3a is mediated by frizzled remains to be investigated.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and antibodies
Fetal calf serum (FCS), glutamine, antibiotics, and trypsin/EDTA were obtained from Life Technologies, Inc. (Basel, Switzerland). {alpha}-MEM was purchased from Amimed (Bioconcept, Allschwill, Switzerland). U0126 and SB202190 were obtained from Calbiochem-Novabiochem Corp. (San Diego, CA). Polyclonal anti-p38, anti-ERK, and anti-β-catenin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Polyclonal anti-pERK, anti-pp38, and anti-pLRP5/6 were obtained from New England BioLabs (Cell Signaling Technology, Beverly, MA). Anti-LRP5/6 was from Biovision (Mountain View, CA). Recombinant mouse SFRP-2 and human SFRP-4 were purchased from R&D Systems (Minneapolis, MN). Recombinant Wnt3a and dickkopf 1 (DKK1) were generous gifts from Novartis AG (Basel, Switzerland) and Prostrakan (Romainville, France), respectively. Recombinant human bone morphogenetic protein-2 (BMP-2) was generously provided by Wyeth Research (Cambridge, MA).

Cell culture and proliferation
Pluripotent mesenchymal C3H10T1/2 cells were cultured in {alpha}-MEM containing 10% FCS (vol/vol), 0.5% nonessential amino acids (vol/vol), 100 IU/ml penicillin, and 100 µg/ml streptomycin. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO2-95% air, and the medium was changed after 48 h. For cell proliferation analysis, cells were seeded at 40,000 cells/ml in 12-well plates. At confluency, they were switched to {alpha}-MEM containing 5% FCS for 24 h. In experiments aimed at testing the effect of inhibitors and their vehicles, cells were preincubated for 4 h before and during experiments with inhibitors. Cell proliferation was determined after 48 h incubation with agents by cell counting (Coulter counter). L cells and Wnt3a-producing L cells were grown to confluency in DMEM and kept for 12 h in 5% FCS for preparation of control and Wnt3a-conditioned medium (cmWnt3a).

ALP activity
ALP activity was determined as previously described (16). Essentially, cells were harvested in 0.2% Nonidet P-40 and disrupted by sonication. The homogenate was centrifuged at 1500 x g for 5 min, and ALP was determined in the supernatant by the method of Lowry et al. (17).

Nodule mineralization and alizarin red staining
For analysis of nodule mineralization, C3H10T1/2 cells were seeded at 40,000 cells/ml in 12-well plates in {alpha}-MEM containing 10% FCS. The day after, they were switched to {alpha}-MEM containing 10% FCS, 10 mM β-glycerophosphate, and 50 µg/ml ascorbic acid (osteogenic medium) as previously described (18, 19). Control medium (20%) and cmWnt3a (20%) were exposed for 10 d with either the MEK inhibitor U0126, the p38 inhibitor SB202190, DKK1, or vehicle. Culture media with or without inhibitors were changed every 2–3 d. Nodule mineralization was analyzed by alizarin red staining as previously described (20). Cell layers were washed twice with cold PBS, fixed with 70% ethanol at –20 C for 1 h and then rinsed once with cold PBS before staining with a 40 mM alizarin red solution in water (Sigma-Aldrich, Basel, Switzerland) for 20 min. Staining was followed by three washes with distilled water and one with 70% ethanol. Nodule mineralization was quantified by extracting the alizarin red stain with 100 nM cetylpyridinium chloride (Sigma-Aldrich) at room temperature for 2 h. Absorbance of the extracted alizarin red stain was measured at 570 nm.

Construction of retroviral vectors and infection of cells
Dominant-negative (dn) p38 tagged with an HA epitope in the 5' end and dnMkk3 and dnMkk6 cDNAs were a generous gift of Dr. J. Han (The Scripp Research Institute, La Jolla, CA). They were cloned into an XhoI site of the pMSCV neo plasmid (Clontech, Palo Alto, CA). For the production of retroviral particles, PT67 packaging cells were transfected with empty pMSCVneo, pMSCVneo-dnp38, or pMSCVneo-dnMkk3 + pMSCVdnMkk6 with Metafecten (Bintex, Martinried/Planegg, Germany). Selection of pMSCV plasmid-expressing cells was obtained after 1 wk culture in 1 mg/ml G418 (Promega, Wallisen, Switzerland). Culture medium from packaging cells containing retroviral particles was filtered and directly added on C3H10T1/2 cells at 70–80% confluency in the presence of 8 µg/ml Polybrene (Sigma-Aldrich) for 24 h. Infected cells were then selected with 1 mg/ml G418 for 1 wk before use for experiments.

Transfections of C3H10T1/2 cells and transient reporter assay
To analyze the β-catenin-driven transcription, subconfluent cells were exposed to 45 µg of the TCF reporter plasmid TOPflash (Upstate Biotechnology, Lake Placid, NY) expressing the firefly luciferase and 9 µg/ml of the control plasmid pRLTX expressing the Renilla luciferase (Clontech, Takara Bio Europ, Saint-Germain-en-Laye, France) with 225 µl polyfect (QIAGEN AG, Hombrechtikon, Switzerland) in 2 ml {alpha}-MEM medium 10% FCS for 18 h. Agents were then added for 24 h before the determination of luciferase activity using the Promega dual-luciferase assay according to the manufacturer’s instructions.

Western blotting analysis
Cell layers of C3H10T1/2 cells treated with different factors or compounds were rapidly frozen in liquid nitrogen and stored at –80 C until used for analysis. For the preparation of lysates, cells were incubated at 4 C in a buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 mM Na3VO4, 1% of the protease inhibitor cocktail Set V (Calbiochem, Merck Ltd., Nottingham, UK), 1% Nonidet P-40, 1% sodium deoxycholate, and 0.1% SDS for 10 min. Lysates were then cleared by centrifugation at 6000 x g for 30 min. For the preparation of the cytosolic fraction, cells were rinsed in PBS and rapidly frozen in liquid nitrogen. They were then scraped in a buffer containing 50 mM Tris (pH 7.4), 0.25 M sucrose, 2 mM Na3VO4, and 1% of the protease inhibitor cocktail Set V and centrifuged at 100,000 x g for 1 h. For the preparation of the nuclear fraction, cells were harvested by scraping and resuspended in hypotonic lysis buffer containing 1 mM EGTA, 1 mM EDTA, 2 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, 10 mM β-glycerophosphate, 2 mM Na3VO4, and 1% of the protease inhibitor cocktail Set V. Cells were swollen for 30 min and broken open with 25 strokes in a tight-fitting Dounce homogenizer. Lysates were layered into tubes containing hypotonic lysis buffer, and spun at 1600 x g for 10 min. The pellet, containing nuclei, was resuspended in an equivalent volume of lysis buffer. A 75-µl sample of either total lysates or cytosolic or nuclear fractions was diluted with an equal volume of 2x reducing sample buffer containing 125 mM Tris buffer (pH 6.8), 4% SDS, 20% glycerol, 0.05% bromophenol blue, and 200 mM dithiothreitol. The mixture was then heated at 70 C for 30 min and subjected to gel electrophoresis on 6–15% gels. After SDS-PAGE, proteins were transferred to Immobilon P membranes and immunoblotted with specific antibodies as previously described (21). Detection was performed using peroxidase-coupled secondary, enhanced chemiluminescence reaction, and visualization by autoradiography (Amersham International, Little Chalfont, UK). Reprobed filters were stripped according to the manufacturer’s protocol. Protein bands were quantified using Image Quant for MacIntosh 1.2 software.

Statistical analysis
All experiments were carried out independently at least three times. Results are expressed as the mean ± SEM. Comparative studies of means were performed using one-way ANOVA followed by a post hoc test (projected least significant difference Fisher) with a significance value of P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
As recently described, Wnt3a stimulated the proliferation and expression of ALP in C3H10T1/2 cells (Fig. 1Go), an in vitro model of mesenchymal cell development into osteoprogenitors (4, 11, 22, 23). The effects of cmWnt3a on cell number (Fig. 1AGo) and ALP activity (Fig. 1BGo) were more pronounced compared with human recombinant Wnt3a. These effects on cell proliferation and differentiation were associated with accumulation of cytosolic β-catenin and activation of MAPKs p38 and ERK (Fig. 2Go). Accumulation of β-catenin in the cytosolic fraction of C3H10T1/2 cells was a slow process. It was detectable only after 2 h with maximal level obtained after 8 h incubation (Fig. 2Go, left panel). In contrast, activation of ERK and p38 was a rapid and transient process that was maximal after 5 min incubation and terminated after 3 h (Fig. 2Go, left panel). Activation of MAPKs and accumulation of β-catenin were also detected in response to recombinant Wnt3a but, as for the cellular effects, activation of these signaling pathways was weaker compared with cmWnt3a (Fig. 2Go, right panel), probably because of loss of peptide activity during the process of purification. To investigate through which receptor molecule Wnt3a induces activation of MAPKs ERK and p38, we used excess (2.5 µg) recombinant secreted frizzled receptor protein 2 and 4 (SFRP-2 and SFRP-4) and DKK1 molecules that are antagonists of Wnt proteins binding to frizzled and LRP5/6, respectively. Whereas DKK1 blunted the activation of LRP5/6 and the accumulation of β-catenin (Fig. 3Go, A and B), both SFRP-2 and -4 failed to interact with this receptor complex (Fig. 3AGo). Activation of p38 and ERK was not affected by DKK1 (Fig. 3AGo), suggesting that LRP5 is not involved in this signaling response. Whether a frizzled molecule is implicated in activation of p38 and ERK remains to be investigated. Associated with the blunting effect of DKK1 on accumulation of β-catenin, the change in ALP activity induced by Wnt3a was markedly reduced by DKK1 (Fig. 3CGo). This LRP5/6 antagonist, however, did not inhibit but rather enhanced ALP stimulation induced by BMP-2 (Fig. 3CGo).


Figure 1
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  		FIG. 1. Effects of Wnt3a on C3H10T1/2 proliferation and expression of ALP. Confluent cells were cultured in 5% FCS containing culture medium for 24 h. A, The number of cells was counted before (T0) and after 48 h treatment with either 20% conditioned medium from L cells culture (Veh), 400 ng/ml recombinant Wnt3a (recWnt), or 20% conditioned medium from L cells stably transfected with cDNA of Wnt3a (cmWnt); B, ALP activity was determined in the same experimental groups after 24 h incubation as described in Materials and Methods. Results are mean ± SEM of four determinations of a representative experiment. *, P < 0.01 compared with vehicle.

 

Figure 2
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  		FIG. 2. Effects of Wnt3a on activation of β-catenin and MAPKs. Confluent C3H10T1/2 cells were cultured in 5% FCS containing culture medium for 24 h. They were then exposed to 20% cmWnt3a for various incubation times (left). They were also incubated with either 20% control conditioned medium (V), 250 ng/ml recombinant Wnt3a (recW), or 20% cmWnt3a (cmW) for 24 h for cytosolic β-catenin (β-cat) and 5 min for p38 and ERK analysis (right). Results are from one representative experiment.

 

Figure 3
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  		FIG. 3. Effect of SFRP2/4 and DKK1 on Wnt3a-induced signaling and stimulation of ALP activity. Confluent C3H10T1/2 cells were cultured in 5% FCS containing culture medium for 24 h. A, Cells were first preincubated with 2.5 µg/ml of SFRP2, SFRP4, or DKK1 for 1 h and then exposed to 20% cmWnt3a (Wnt3a) or 20% control conditioned medium (Veh) for 5 min (p38 and ERK) or 6 h (LRP5 and β-catenin) before analysis of signaling proteins in total cell lysates by Western blot; B, cells were preincubated with 1 µg/ml DKK1 for 1 h and then exposed to 20% cmWnt3a (Wnt3a) or control conditioned medium for 24 h before analysis of β-catenin accumulation in the cytosolic fraction; C, confluent cells were exposed to 20% control conditioned medium (Veh), 20% cmWnt3a (Wnt3a), or 50 ng/ml BMP-2 in presence or absence of DKK1 for 24 h before determination of ALP activity. *, P < 0.01 compared with vehicle; §, P < 0.001 compared with the Wnt3a-treated group.

 
The observation that Wnt3a can also activate p38 in pluripotent mesenchymal cells in addition to the canonical β-catenin pathway (24, 25) and ERK (14, 15) was of potential interest because this MAPK has been reported to play an important role in the differentiation of osteoblastic cells (16, 26, 27, 28, 29). Thus, we analyzed the respective role of ERK and p38 for Wnt3a-induced changes in C3H10T1/2 cell proliferation and differentiation. As shown in Fig. 4Go, A and B, the presence of a selective inhibitor of the ERK pathway completely blunted cell growth and markedly reduced expression of ALP induced by Wnt3a, suggesting an important role of this pathway for Wnt-induced mesenchymal cell development. Interestingly, the presence of a selective p38 inhibitor did not influence cell growth induced by Wnt3a; if anything, it slightly enhanced this response (Fig. 4AGo). However, it nearly completely blunted the stimulation of ALP induced by Wnt3a (Fig. 4BGo). Associated with this effect, this inhibitor also completely blunted nodule mineralization induced by Wnt3a in cells cultured in an osteogenic medium, whereas the selective MEK inhibitor had no effect and DKK1 slightly reduced this response (Fig. 4CGo). This observation strongly suggests that p38 is an important signaling pathway for controlling ALP expression and matrix mineralization induced by Wnt3a in C3H10T1/2 cells cultured in an osteogenic medium. To validate the above observations obtained with the specific p38 inhibitor, we constructed two series of C3H10T1/2 cells expressing either inactive p38 (dnp38) or inactive MAPK kinases (MKK) 3 and 6 (dnMKK3/6) molecules using a retroviral system. One representative group of cells infected with each dominant molecule is presented in Fig. 5Go. Clearly, expression of dn molecules of the p38 pathway markedly reduced both baseline and stimulated ALP induced by Wnt3a by 63–80% (results of six different preparations of infected cells). The lower ALP activity in clones expressing the dn molecules was not due to a nonspecific alteration in activation of this gene because BMP-2 was able to stimulate ALP activity at levels even higher than that recorded in cells exposed to Wnt3a and stably infected with the empty vector (Fig. 5Go). Increased cell number induced by Wnt3a was also not affected by dnp38 and dnMKK3/6 expression (2.31 ± 0.04- and 2.49 ± 0.10-fold, respectively) compared with empty vector-expressing cells (2.33 ± 0.06-fold). These observations indicate that the p38 pathway is an important determinant of Wnt-induced mesenchymal cell differentiation into skeletogenic cells. To assess whether p38 influences the canonical β-catenin pathway, we measured changes in Wnt3a-induced β-catenin transcriptional activity using the TOPflash TCF reporter and pRLTK plasmids. Results shown in Fig. 6AGo indicate that β-catenin transcriptional activity is markedly and dose-dependently decreased by the p38 inhibitor. It was also decreased in cells expressing dnp38 and dnMKK3/6 (Fig. 6BGo) but not by the inhibitor of the ERK pathway (6C). In comparison and as expected, DKK1 (5 µg/ml) completely blunted β-catenin transcriptional activity (Fig. 6DGo). Because p38 is activated at a much earlier time point than accumulation of β-catenin (Fig. 2Go), we investigated whether this MAPK interacts with the process of β-catenin stabilization. Apparently, p38 does not influence activation of the canonical β-catenin pathway because accumulation of β-catenin in the cytosol was not affected by either the p38 and the MEK inhibitor (Fig. 7AGo) or by expression of dnp38 or dnMKK3/6 molecules (Fig. 7BGo). Accumulation of β-catenin induced by Wnt3a in the nucleus was also not affected by the p38 inhibitor (Fig. 7CGo). These observations suggest that the effect of p38 on β-catenin-induced transcriptional activity (Fig. 6AGo) is downstream activation of β-catenin, possibly at the transcriptional level. Thus, there was no apparent interaction between early activation steps in β-catenin and MAPKs p38 and ERK pathways triggered by Wnt3a in C3H10T1/2 cells.


Figure 4
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  		FIG. 4. Effects of MAPK inhibitors on cell proliferation, ALP activity, and nodule mineralization induced by Wnt3a. Confluent C3H10T1/2 cells were cultured in 5% FCS containing culture medium for 24 h. They were first preincubated with 10 µM of the MEK1,2 inhibitor U0126 (U0), 10 µM of the p38 inhibitor SB202190 (SB), or vehicle (Veh) for 4 h. Then they were exposed to 20% of either cmWnt3a (Wnt3a) or control conditioned medium. A and B, Cell number (A) was determined before (T0) and after 48 h incubation, whereas ALP activity (B) was determined after 24 h incubation; C, at confluency, C3H10T1/2 cells were cultured in an osteogenic medium containing 10% FCS, 10 mM β-glycerophosphate, and 50 µg/ml ascorbic acid. Cells were preincubated for 4 h with or without U0126 (10 µg/ml), SB202190 (SB, 10 µg/ml), and DKK1 (1 µg/ml) before exposure to either 20% cmWnt3a (Wnt3a) or control medium (Veh). Culture medium containing the different agents was changed every 2–3 d. After 10 d culture, cells were fixed in 70% ethanol and stained with alizarin red for the determination of nodule mineralization as described in Materials and Methods. Results are mean ± SEM of four determinations of a representative experiment. +, P < 0.05; *, P < 0.01 compared with Veh; §, P < 0.01 compared with the Wnt3a-treated group.

 

Figure 5
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  		FIG. 5. Effects of dn molecules of the p38 pathway on ALP activity induced by Wnt3a and BMP-2. C3H10T1/2 cells were infected with the pMSCVneo retroviral vector containing either dnp38 cDNA or dnMKK3 and dnMKK6 cDNA (dnMek3/6) or with the empty vector (Neo). Then, they were stimulated with either 20% cmWnt3a (Wnt3a) or 100 ng/ml BMP-2 for 24 h before the determination of ALP activity. Results are mean ± SEM of four determinations of a representative experiment. *, P < 0.01 compared with Veh; §, P < 0.001 compared with the respective Neo-treated group.

 

Figure 6
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  		FIG. 6. Effects of various inhibitors on Wnt3a-induced β-catenin transcriptional activity in C3H10T1/2 cells. Subconfluent cells were transfected with TOPflash firefly and pRLTK renilla plasmids for 24 h and preincubated for 4 h with or without various concentrations of SB202190 (SB202) (A), U0126 (C), 5 µg/ml DKK1 (D), or vehicle. Stably infected cells with empty pMSCV vector (neo) or with pMSCV-dnp38 (dnp38) or pMSCV-dnMkk3+Mkk6 (dnMKK3/6) were transfected with TOPflash firefly and pRLTK renilla plasmids for 24 h (B). Then all cells were exposed to cmWnt3a (Wnt3a) for 16 h before the determination of β-catenin transcriptional activity. Data are expressed as fold increase firefly corrected by renilla luciferase by Wnt3a compared with control. Results are mean ± SEM of four determinations of one representative experiment for each study. §, P < 0.001 compared with Wnt3a-treated group; *, P < 0.05 compared with the neo group with Wnt3a.

 

Figure 7
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  		FIG. 7. Effects of MAPK inhibitors on Wnt3a-induced cytosolic and nuclear β-catenin accumulation. Confluent C3H10T1/2 cells were cultured in 5% FCS containing culture medium for 24 h. A, Cells were first preincubated with various concentrations of the p38 inhibitor SB202190 (SB202), 10 µM of the MEK1,2 inhibitor U0126, or vehicle for 4 h; B, C3H10T1/2 cells were infected with the pMSCVneo retroviral vector containing either dnp38 cDNA or dnMKK3 and dnMKK6 cDNA (dnMek3/6) or with the empty vector (Neo). Then they were exposed to 20% of either cmWnt3a (Wnt3a) or control conditioned medium. Cytosolic (A and B) and nuclear (C) fractions were then isolated as described in Materials and Methods. Total β-catenin accumulation was determined by Western blot analysis as described in Materials and Methods using specific antibodies. Results are from one representative experiment.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Two recent studies described that Wnt proteins can activate the ERK and PI3K pathways and regulate the proliferation of fibroblasts and apoptosis of osteoblastic cells. In fibroblasts, Wnt3a activated the ERK pathway via the Ras->Raf-1->MEK->ERK cascade (14), whereas in osteoblastic cells, a Src-dependent and LRP5/6-independent mechanism has been described (15). Results of these studies indicated that in mammalian cells, Wnt proteins activate not only the β-catenin pathway but also other pathways such as ERK and PI3K that contribute to the cellular responses induced by Wnts. It has recently been shown that the canonical Wnt-β-catenin pathway plays an important role in osteoblastogenesis for the differentiation of osteoblasts from a precursor that is shared with the chondrocyte lineage and for the development of osteoblasts during postnatal life in bone homeostasis (reviewed by Hartmann in Ref. 30). Essentially, it has been proposed that cellular β-catenin concentrations have to be elevated to enable differentiation into osteoblasts, whereas levels must be low for differentiation of precursors into the chondrocyte lineage (12, 13). Other pathways have been shown to be involved in the differentiation of mesenchymal cells into osteoblasts such as Hedghog and BMPs (10, 11, 31, 32). In the present study, we investigated whether Wnt proteins can activate different MAPKs and their functional role in mesenchymal cells that have the potential to differentiate into osteoprogenitors in presence of Wnt3a (18, 23). First we confirmed that Wnt3a increases the proliferation, ALP expression, and nodule mineralization in C3H10T1/2 cells (18, 23). We also found and further documented that Wnt3a activates both the canonical β-catenin pathway and ERK as recently described (14, 15, 23). In addition to activation of β-catenin and ERK, we discovered that p38 is also activated by Wnt3a in C3H10T1/2 cells. To avoid nonspecific signaling effects by factors other than Wnt3a in the conditioned medium from L cells, we also analyzed signaling responses induced by the recombinant Wnt3a peptide. Identical but lower signaling and cellular responses were observed with the recombinant protein compared with the cmWnt3a, probably because of some loss of activity during peptide purification. A striking difference in the kinetics of activation between the canonical β-catenin and MAPK pathways was noted (Fig. 2Go). Whereas phosphorylation of ERK and p38 was rapid and transient, accumulation of β-catenin in the cytosol was relatively slow, being maximal after 8 h incubation and maintained for several hours (Fig. 2Go). Rapid MAPK activation is probably mediated by the direct stimulation of a sequential protein phosphorylation cascade (33), whereas accumulation of β-catenin results from inhibition of the ubiquitin-proteasome system (34). It is now well documented that Wnt ligands initiate signaling by interacting with two types of receptor molecules at the cell membrane, frizzled and LRP5/6. Wnt antagonists play important roles in regulating Wnt signaling. With the exception of DKKs, most of the other known families of secreted antagonists, such as Cerberus and Fzb, function by binding extracellular Wnt ligands, thus preventing them from interacting with frizzled receptors (35). Secreted SFRP inhibition of Wnt signaling may operate through at least two mechanisms: 1) by competition with frizzled for Wnt ligands or 2) in a dn fashion by direct formation of nonsignaling inactive complexes with frizzled receptors (36). In contrast, the DKK family of proteins antagonizes Wnt signaling by acting as an inhibitory ligand of LRP5/6 (37, 38, 39). Evidence has been provided that DKK blocks the β-catenin pathway but leaves the cells free to respond via the planar polarity pathway mediated by frizzled receptors (38, 40). To assess which receptor molecule is involved in activation of p38, we studied the effects of SFRP-2 and SFRP-4 as well as DKK1 on β-catenin and MAPK signaling. As recently described in osteoblasts (41), DKK1 blunted the LRP5/6-induced β-catenin pathway in C3H10T1/2 cells (Fig. 3Go, A and B) but had no effect on activation of ERK and p38 induced by Wnt3a (Fig. 3AGo). Both SFRP-2 and SFRP-4 did not antagonize activation of ERK, p38, and LRP5/6 by Wnt3a. This observation suggests that they did not compete with Wnt3a binding to LRP/frizzled receptors. We have chosen these antagonists because they were recently shown to counteract Wnt3a effects in MC3T3-E1 cells (42) and L cells (43). The difficulty in observing an antagonistic effect of SFRPs on Wnt signaling has also been observed in other reports and is probably explained by the complex mechanism involved for the Wnt antagonistic function of these molecules (44). Thus, it remains unclear whether a frizzled receptor is involved in mediating the activation of MAPKs ERK and p38 by Wnt3a in C3H10T1/2 cells as previously reported for the c-Jun N-terminal kinase (JNK) planar polarity pathway. Whether dishevelled (Dsh/Dvl), which mediates activation of JNK in response to Wnt proteins (45, 46, 47, 48, 49), is also involved in the stimulation of ERK and p38 remains to be investigated.

The use of the specific MEK inhibitor U0126 confirmed that ERK influences cell proliferation induced by Wnt3a in mammalian cells (14). We found that this pathway is probably also involved in the commitment of mesenchymal cells into skeletogenic cells because it markedly reduced the stimulation of ALP induced by Wnt3a (Fig. 3BGo). The p38 inhibitor also markedly decreased this response (Fig. 3BGo). However, in contrast to the MEK inhibitor, it did not influence the mitogenic effect of Wnt3a (Fig. 3AGo). Its effect on ALP was therefore growth independent. In addition to influencing expression of ALP, the p38 inhibitor also blunted nodule mineralization induced by Wnt3a in C3H10T1/2 cells cultured in an osteogenic medium (Fig. 3CGo), strongly suggesting an important role of the p38 pathway in the differentiation of mesenchymal cells by Wnt3a. Interestingly, the MEK inhibitor did not influence the Wnt3a-induced nodule mineralization response but markedly affected basal matrix mineralization of C3H10T1/2 cells (Fig. 3CGo). This observation confirms recent information obtained in MLO-A5 and MC3T3-E1 cells indicating that the ERK pathway negatively regulates matrix mineralization (50). Also of interest was the relatively modest effect of DKK1 on nodule mineralization induced by Wnt3a compared with the p38 inhibitor (Fig. 4CGo), suggesting that the β-catenin pathway may not be the principal pathway for controlling matrix mineralization induced by Wnt proteins in osteoprogenitors. A markedly reduced basal and stimulated ALP activity by Wnt3a was also recorded in cells stably transfected with either dnp38 or dnMKK3/6, further suggesting an important role of p38 in Wnt3a-induced mesenchymal cell differentiation. Interestingly, expression of either dnp38 or dnMKK3/6 in C3H10T1/2 cells did not affect the stimulation of ALP induced by BMP-2 (Fig. 1Go), suggesting a minor role of p38 in BMP-2-induced differentiation of C3H10T1/2 cells. The observation that p38 is involved in mesenchymal cell differentiation induced by Wnts was of potential interest because we and another laboratory described that p38 is also involved in the differentiation of osteoblastic cells, essentially in expression of ALP and matrix mineralization (16, 27, 28). The molecular mechanism by which p38 regulates ALP and other parameters of osteogenic cell differentiation remains poorly defined. Because the canonical β-catenin pathway is a potent inducer of the differentiation of osteoprogenitors, we explored whether p38 interacts with the β-catenin pathway. The TOPflash reporter system was used to assess this relationship. As previously described, Wnt3a induced a strong transcriptional activity associated with β-catenin accumulation in C3H10T1/2 cells (15, 18). This activity was dose-dependently and markedly reduced in the presence of the p38 inhibitor (Fig. 6AGo). It was also significantly reduced in cells infected with dnp38 and dnMKK3/6 retroviral vectors (Fig. 6BGo) and by DKK1 (Fig. 6DGo) but not in presence of the MEK inhibitor U0126 (Fig. 6CGo). These observations strongly suggest that p38 interacts with the β-catenin pathway. To assess whether this interaction takes place early in the activation steps of the Wnt/β-catenin pathway, we measured accumulation of cytosolic β-catenin in cells exposed to the p38 inhibitor or in cells expressing either dnp38 or dnMKK3/6. No change in the amount of β-catenin could be recorded in a dose-inhibitory range of the p38 inhibitor (Fig. 7AGo) that clearly decreased the β-catenin transcriptional activity induced by Wnt3a (Fig. 6AGo). There was also no reduction in accumulation of cytosolic β-catenin in cells infected with the dnp38 and dnMKK3/6 plasmids. Because it has been reported that p38 can regulate import/export of various molecules (51), we also measured nuclear accumulation of β-catenin in cells exposed to Wnt3a with and without the p38 inhibitor. No effect of the p38 inhibitor was found on Wnt3a-induced β-catenin nuclear accumulation (Fig. 7CGo). Thus, these observations strongly suggest that p38 interacts with the β-catenin pathway downstream of the activation steps, probably at the transcriptional level by a molecular mechanism that remains to be investigated. Recent studies in osteoblastic cells described interactions between signaling induced by Wnts and BMPs. It was first described that the β-catenin pathway participates in BMP-2 mediated signal transduction (23). It was then reported that the capacity of BMP-2 to induce ALP in bone cells relies on Wnt expression (22). More recently, a potential mechanism whereby BMP-2 antagonizes Wnt signaling in osteoblast progenitors by promoting an interaction between Smad1 and Dvl-1 that restricts β-catenin activation has been described (52). In our analyses of the effect of DKK1 on Wnt3a-induced ALP expression in C3H10T1/2 cells (Fig. 3CGo), we also investigated the influence of DKK1 on BMP-2-induced ALP expression. Inhibition of the β-catenin pathway by DKK1 did not prevent the differentiating effect of BMP-2. In fact, inhibition of the β-catenin pathway was associated with an even higher stimulation of ALP (Fig. 3CGo), indicating that the Wnt/β-catenin pathway is probably not necessarily required for the BMP-2 effect on the differentiation of these cells into skeletogenic cells. Our observation is rather consistent with the existence of an interaction between Smad1 and Dvl-1 (52) that restricts both BMP and Wnt signaling in osteoprogenitors.

Finally, a role of a p38 kinase in the planar polarity pathway, which would be redundant with JNK, has been suggested in Drosophila (53). This information and our report in mesenchymal cells suggest that activation of p38 by Wnt receptors might be a conserved signaling pathway in various species.

In conclusion, data provided in this study indicate that, in addition to activation of the canonical β-catenin and of the ERK pathway, Wnt proteins can also activate p38. They also suggest that this MAPK plays an important role in the differentiation of mesenchymal cells into osteoprogenitors. Activation of p38 by Wnt receptors is independent of LRP5/6. Whether activation of this pathway involves a frizzled molecule remains to be investigated.


   Acknowledgments
 
We thank P. Apostolides for his expert technical assistance.


   Footnotes
 
This study was supported by the Swiss National Science Foundation (3100AO-112146).

Disclosure Statement: The authors of this manuscript have nothing to declare.

First Published Online August 23, 2007

Abbreviations: ALP, Alkaline phosphatase; BMP, bone morphogenetic protein; cmWnt3a, Wnt3a-conditioned medium; DKK, dickkopf; dn, dominant-negative; FCS, fetal calf serum; JNK, c-Jun N-terminal kinase; LRP, low-density lipoprotein-receptor-related protein; MKK or MEK, MAPK kinase; PI3K, phosphatidylinositol 3-kinase; SFRP, secreted frizzled receptor protein.

Received April 20, 2007.

Accepted for publication August 10, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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