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Originally published In Press as doi:10.1074/jbc.M602308200 on August 14, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31720-31728, October 20, 2006
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Wnt/beta-Catenin Signaling Is a Normal Physiological Response to Mechanical Loading in Bone*

John A. Robinson{ddagger}21, Moitreyee Chatterjee-Kishore§2, Paul J. Yaworsky§, Diane M. Cullen, Weiguang Zhao{ddagger}3, Christine Li§, Yogendra Kharode{ddagger}, Linda Sauter§4, Philip Babij§5, Eugene L. Brown§, Andrew A. Hill§, Mohammed P. Akhter, Mark L. Johnson6, Robert R. Recker, Barry S. Komm{ddagger}, and Frederick J. Bex{ddagger}

From the {ddagger}Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, Pennsylvania 19426, §Biological Technologies, Wyeth Research, Cambridge, Massachusetts 02140, and Creighton University, Osteoporosis Research Center, Omaha, Nebraska 68131

Received for publication, March 10, 2006 , and in revised form, July 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A preliminary expression profiling analysis of osteoblasts derived from tibia explants of the high bone mass LRP5 G171V transgenic mice demonstrated increased expression of canonical Wnt pathway and Wnt/beta-catenin target genes compared with non-transgenic explant derived osteoblasts. Therefore, expression of Wnt/beta-catenin target genes were monitored after in vivo loading of the tibia of LRP5 G171V transgenic mice compared with non-transgenic mice. Loading resulted in the increased expression of Wnt pathway and Wnt/beta-catenin target genes including Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43 in both genotypes; however, there was a further increased in transcriptional response with the LRP5 G171V transgenic mice. Similar increases in the expression of these genes (except cyclin D1) were observed when non-transgenic mice were pharmacologically treated with a canonical Wnt pathway activator, glycogen synthase kinase 3beta inhibitor and then subjected to load. These in vivo results were further corroborated by in vitro mechanical loading experiments in which MC3T3-E1 osteoblastic cells were subjected to 3400 microstrain alone for 5 h, which increased the expression of Wnt10B, SFRP1, cyclin D1, FzD2, WISP2, and connexin 43. Furthermore, when MC3T3-E1 cells were treated with either glycogen synthase kinase 3beta inhibitor or Wnt3A to activate Wnt signaling and then subjected to load, a synergistic up-regulation of these genes was observed compared with vehicle-treated cells. Collectively, the in vivo and in vitro mechanical loading results support that Wnt/beta-catenin signaling is a normal physiological response to load and that activation of the Wnt/beta-catenin pathway enhances the sensitivity of osteoblasts/osteocytes to mechanical loading.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Over the past few years the Wnt/beta-catenin-signaling pathway has been shown to be an important component of bone mass accrual, regulation, and maintenance. Key to this understanding were the identification of inactivating mutations in LRP5 resulting in an osteoporosis pseudoglioma syndrome (1) and gain of function mutations in the LRP5 gene that give rise to a high bone mass phenotype in humans (2-4). Both of these conditions can be mimicked in mice (5, 6).

LRP5 and LRP6 have been shown to be co-receptors (7-10) with the frizzled family of receptors and are involved in signaling through the canonical Wnt/beta-catenin pathway (11, 12). Upon binding of extracellular Wnt to LRP5/6 and frizzled coreceptor complex, dishevelled (Dsh) is activated, causing axin recruitment to the membrane (where it interacts with LRP5/6) and inactivation of glycogen synthase kinase-3beta (GSK-3beta)7 that results in stabilization and accumulation of beta-catenin in the cytoplasm (13). Stabilized beta-catenin translocates to the nucleus and together with T-cell factor/lymphoid enhancer binding factor transcription factors activates transcription (12-14). Regulation of GSK-3beta is a key step in the pathway, and inhibitors of this enzyme can lead to beta-catenin stabilization and initiation of target gene expression independent of Wnt binding. Signaling through this pathway is also regulated at the extracellular level by a number of modulator proteins (15-22).

We have found that the increased peak bone mass that results in mice overexpressing the high bone mass (HBM) mutated gene product (LRP5 G171V) is due primarily to osteoblast-induced positive bone balance (5). The bone mineral density in these animals plateaus and is maintained through 1.5 years of age without apparent abnormalities in bone shape or size. Furthermore, the LRP5 G171V mutation in mice results in a skeleton that has enhanced structural strength (femur, femoral neck, tibiae, and vertebral body), material properties (vertebral body), and bone mass/ash weight (ulnae) than their non-transgenic littermates (23).

The denser and stronger bones in LRP5 G171V transgenic mice have been suggested to be due to greater sensitivity of bone to normal mechanical stimuli resulting in an altered response to weight-related forces (23). This pattern is quite similar to that seen clinically in humans where affected members have increased bone mineral density and bones that are of normal shape and size. In the LRP5 G171V HBM kindred the most significant changes in bone density are associated with the load bearing bones (24, 25). Furthermore, we have shown that HBM transgenic mice (5) have increased sensitivity to mechanical load (25, 26), whereas Sawakami et al. (27) have shown in vivo that bone formation response as a result of loading of LRP5-/- mice was nearly abolished compared with wild type mice. In addition, Hens et al. (28) have shown the activation of the Wnt/beta-catenin-signaling pathway in osteoblasts from TOPGAL mice containing a lacZ gene driven by a T-cell factor promoter. Finally, Clement-Lacroix et al. (29) have shown that the GSK-3beta inhibitor LiCl increases bone formation in LRP5-/- mice. Collectively, these data strongly suggest that LRP5 and the Wnt/beta-catenin-signaling pathway play a critical role in the bone response to mechanical loading.

Although the molecular mechanisms by which mechanical loading affects bone mineral density have not been fully elucidated, various in vitro and in vivo models of mechanical loading have attributed it to increased cell proliferation, activation of cell signaling, and transcriptional activation of a number of genes. Rapid signaling responses have also been reported including changes in intracellular Ca+2, release of prostaglandins (PGE2 and PGI2), nitric oxide release, and increases in cAMP levels. (30-35). The studies described in this report demonstrate that a number of target genes of the Wnt/beta-catenin-signaling pathway are up-regulated in response to mechanical loading and that the LRP5 G171V mutation results in exaggerated increases in expression of several of these genes. We further show that activation of the pathway by treatment with a GSK-3beta inhibitor results in an anabolic bone formation response and that use of this inhibitor in combination with mechanical loading produces a synergistic effect on the expression of Wnt/beta-catenin pathway target genes. These results strongly implicate the Wnt/beta-catenin-signaling pathway as being a critical component of the bone response to mechanical loading.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Calcein, hematoxylin, and eosin were obtained from Sigma-Aldrich. Fetal bovine serum (FBS), heat-inactivated FBS, {alpha}-minimum essential medium, Dulbecco's modified Eagle's medium, penicillin, streptomycin, L-glutamine, glutamax, and Geneticin were purchased from Invitrogen. Bovine serum albumin was purchased from Serologicals Proteins Inc. (Kankakee, IL).

In Vivo Loading of Tibiae in LRP5 G171V Transgenic and Non-transgenic Littermates—All animal protocols were conducted with approval of the Wyeth and Creighton University Institutional Animal Care and Use Committees. The heterozygous LRP5 G171V transgenic mice have been described and show a statistically significant increase in bone density (5). Non-transgenic littermates were used as controls. There were a total of 15 animals/sex/genotype in each group. At 17 weeks of age all animals were anesthetized to permit proper leg positioning before loading. Using a 4-point bending device (23), the mechanical loading regimen (~2500 microstrain)8 delivered to the right tibiae (the left tibiae served as the non-loaded controls) was 6N for females and 7N for males (36 cycles, 2 Hz), which ensured that all mice experienced similar levels of maximal compressive and tensile strains during bending loads. RNA from the right tibiae was collected at 4 or 24 h after application of load. Tibiae from 5 mice were pooled to compose a single group. Three replicate groups for each treatment/genotype were analyzed.

Cell Culture—MC3T3-E1 osteoblastic cells, used in the in vitro mechanical loading experiments, were cultured in {alpha}MEM supplemented with 10% heat inactivated fetal bovine serum, 1% glutamax, and 1% penicillin/streptomycin. Wnt3A-conditioned media was obtained from an overexpressing Wnt3A stable murine L-cell line (ATCC, Manassas, VA) that was maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% L-glutamine, and 0.4 mg/ml Geneticin. To obtain Wnt3A-conditioned media, cells were seeded into 100-mm dishes and cultured for 4 days in growth medium without Geneticin, the medium was removed and sterile-filtered, and fresh medium was added to the plates and cultured for an additional 3 days. The medium was then removed and sterile-filtered and combined with the initial batch of cultured media. Control-conditioned medium was obtained in a similar fashion using the parental L-M(TK-) cell line (ATCC, Manassas, VA). The Wnt3A-conditioned media activated canonical Wnt signaling in MC3T3-E1 cells as determined using a T-cell factor-luciferase reporter transiently transfected into these cells (10 µl of conditioned media showed a 10-fold induction of reporter activity compared with control media-treated and untreated cells (data not shown).

RNA Isolation—The mouse tibiae were dissected free of soft tissue, and the proximal and distal metaphysis were removed leaving the diaphysis. The tibiae were then cut transversely with bone cutters to expose the trabecular bone and the bone marrow. The trabecular bone and marrow cavities were flushed with ice-cold sterile phosphate-buffered saline to remove the marrow, and the clean bone was placed in liquid nitrogen. A Bessman tissue pulverizer (Fisher) rinsed in 100% ethanol and precooled in liquid nitrogen was used to reduce the tibiae to a powder. Total RNA (2 µg/tibia) was isolated from non-loaded and loaded bones using the ToTALLY RNA kit (Ambion, Austin TX) as per the manufacturer's instructions. Ten µg of total RNA was treated with 4 units of DNase I (Ambion) to remove any genomic DNA contamination.

To isolate RNA from MC3T3-E1 cells the cultures were washed twice with 2 ml each of phosphate-buffered saline, and then the RNA was isolated using the QIAshredder and the RNeasy kit (Qiagen, Valencia, CA) as described by the manufacturer. The RNA was treated with 27 units of DNase I (Qiagen) on the RNA isolation column provided in the kit as described by the manufacturer.

Quantitative Real-time RT-PCR (TaqMan®)—A two-step TaqMan protocol was used. RNA was first converted to cDNA at 37 °C for 2 h (High Capacity cDNA Archive kit, Applied Biosystems). TaqMan PCR reactions were performed on an ABI Prism 7700 DNA sequence detector (Applied Biosystems) using 20 ng of cDNA/reaction. The conditions for TaqMan PCR were 2 min at 50 °C, 10 min at 95 °C, then 40 cycles each of 15 s at 95 °C and 1 min at 60 °C on MicroAmp Optical 96-well plates covered with MicroAmp Optical caps. Each plate contained triplicates of the test cDNA templates and no-template controls for each reaction mix. The expression for each mouse gene was normalized to murine glyceraldehyde-3-phosphate dehydrogenase. A list of TaqMan probe-primer pairs used in this study can be found in Table 1.


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  		TABLE 1
TaqMan primer and probe sequences

Ptgis, prostacyclin synthase.

 
Calvariae Treatment and Histochemistry and Immunohistochemistry Analysis—The GSK-3beta inhibitor (GSK3betai) (3-(3-chloro-4-hydroxyphenylamino)-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione) (36) at 1 mg/kg or vehicle was injected subcutaneously daily for the first 7 days over the right side of the calvaria in 4-week-old male Swiss-Webster mice. For each treatment group that contained 8 animals each, calcein (15 mg/kg) was administered subcutaneously on day 1 and again on day 8 for mineral apposition rate (MAR) determination. On day 10 the calvariae were removed, and the bones were fixed in 70% ethanol for 24 h. The anterior portion of the calvaria was paraffin-embedded, whereas the posterior portion was used for frozen sections. The paraffin sections were stained with hematoxylin and eosin for measurement of calvarial thickness.

To calculate mineral apposition rates linear measurements of single label surface (SLS), double label surface (DLS), and bone surface (BS) were taken, and the equation DLS + (1/2 SLS)/BS x 100 was used to calculate percent mineralized surface/bone surface. Measurements were made on unstained 6-µm sections at 20x magnification. All measurements were made using the Bioquant Image Analysis System (Bioquant, Nashville, TN). For immunohistochemical analysis of beta-catenin, calvaria were decalcified in Surgipath Decalcifier II (Surgipath, Richmond, IL) for 7-8 h, dehydrated in graded alcohol, and sectioned. Nonphosphorylated beta-catenin was detected using a mouse monoclonal antibody (Upstate%20Biotechnology">Upstate Biotechnology, Lake Placid, NY). Signal was detected using the avidin-linked AP system (Vector Laboratories, Burlingame, CA). Endogenous alkaline phosphatase was detected histochemically using the Vector Red alkaline phosphatase substrate kit (Vector Laboratories) in 6-µm frozen sections of the mouse parietal bone after fixation in 70% ethanol.

Effect of Systemic GSK3betai Administration on the in Vivo Response to Mechanical Load—17-Week old female, wild type C57Bl6 mice were injected with GSK3betai 50 mg/kg/BID or vehicle (control) subcutaneously twice daily for 14 days. There were 15 animals per group. The right tibiae were loaded at 6N for 36 cycles at 2 Hz. The left tibiae served as unloaded controls. The animals were sacrificed at 4 h post-load, and the tibia was isolated and flash-frozen in liquid nitrogen. Tibiae (five) were pooled from each group to provide three replicates per group. The RNA was purified from tibiae (loaded and unloaded). Transcriptional analyses were performed by TaqMan® on samples from the tibiae on selected load- and Wnt-response genes.

In Vitro Mechanical Loading—MC3T3-E1 cells were plated at 80,000-100,000 cells per well in a type I collagen-coated Bioflex 6-well plate (Flexcell International Corp., McKeesport, PA) and then cultured for 3-4 days or until confluent. Twenty-four hours before loading the cells were washed twice with 2 ml of basal {alpha}-minimum essential medium ({alpha}MEM) before adding 2 ml of fresh serum-free media containing {alpha}MEM, 0.25% bovine serum albumin, glutamax, and penicillin/streptomycin. Immediately before mechanical loading, the medium was removed, and 1 ml of {alpha}-minimum essential medium/bovine serum albumin with or without GSK3betai, Wnt3A-conditioned media or control-conditioned media was added to each well. The cells were subjected to mechanical distortion equivalent to 3400 microstrain (2 Hz, 7200 cycles/h) for 5 h using a FX-3000 Flexercell® strain unit (Flexcell International Corp). RNA was harvested immediately or 24 h post-loading from both the mechanical-strained samples as well as the non-strained controls.

Statistical Analysis—The data are represented as the mean ± S.D. For those data comparing non-loaded versus loaded results, unpaired 2-tailed t-tests was performed. To compare strain and GSK-3beta inhibitor or Wnt3A, multiple comparisons of 2-factor analysis of variance was performed. A Tukey HSD multiple comparisons was then performed between each dose of the GSK-3beta inhibitor or Wnt3A with strain versus the strain only treatment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Application of in Vivo Mechanical Load Induces Transcription of Wnt/beta-Catenin Pathway Genes—It has been previously reported that the LRP5 G171V transgenic mice have increased bone formation compared with non-transgenic mice (5), and mechanical loading increases bone formation. Given the fact that this mutation leads to altered regulation of the Wnt/beta-catenin pathway, we tested the hypothesis that downstream targets of the pathway would have exaggerated changes in expression in response to loading compared with normal mice. We performed a preliminary analysis of gene expression profiles from tibial osteoblast explant cultures from LRP5 G171V versus non-transgenic mice and identified several differentially expressed genes including Wnt10B, secreted frizzled-related protein 1 (SFRP1), SFRP2, Dickkopf-3 (Dkk3), cyclin D1 (CCND1), and Wnt1-inducible signaling pathway protein 2 (WISP2) associated with the mutation (data not shown). We next examined whether the expression of these Wnt/beta-catenin target genes were regulated by mechanical loading. Transcriptional analysis of tibia samples from LRP5 G171V mice after in vivo mechanical loading by four-point bending resulted in expected increases in the mRNA expression of known stress-responsive genes including prostaglandin synthase (COX-2), prostacyclin synthase (Ptgis), and endothelial nitric-oxide synthase (eNOS) (37, 38) (Fig. 1). The expression of these genes was increased 4 h post-loading in wild type and LRP5 G171V transgenic mice tibiae; however, the response was 4-10-fold greater in the transgenics (Fig. 1). This response was attenuated 24 h post-loading (data not shown).


Figure 1
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  		FIGURE 1.
Expression analysis of known stress responsive genes in LRP5 G171V transgenic (TG) and non-transgenic mice subjected to mechanical load. Quantitative RT-PCR analysis from both male and female tibia RNA shows mRNA levels for COX2, prostacyclin synthase (Ptgis), and eNOS genes 4 h after loading. Transcriptional responses are represented as -fold change (Log2) of loaded responses relative to non-loaded sample responses (*, p < 0.01).

 
In non-transgenic mice, transcription of Wnt10b, SFRP1, CCND1, connexin 43 (Cxn43), and WISP2 genes was significantly increased (~2-4-fold, p < 0.01) at both 4 and 24 h after in vivo mechanical loading (Fig. 2, A and B). RNA levels of frizzled 2 (FzD2) was increased 2.8-fold (p < 0.01) at 4 h post-load but returned to base line by 24 h. Expression of LRP5 was not affected by load in this model, whereas LRP6 expression was marginally increased in male LRP5 G171V animals at 4 h post-load (Fig. 2, A, and B). In LRP5 G171V transgenic mice, a more significant and sustained increase (3-30-fold, p < 0.01) in the transcription of the Wnt/beta-catenin target genes analyzed was observed, including SFRP4, which was not regulated by mechanical loading in wild type mice (Fig. 2, A and B). Unlike the pattern demonstrated in the non-transgenic mice, there was an increased expression of FzD2 at both 4- and 24-h post-load. Thus, application of mechanical load increased expression of Wnt/beta-catenin-regulated genes, and the response was significantly greater in the LRP5 G171V transgenic mice.

Activation of Wnt/beta-Catenin Signaling and Bone Formation by an Inhibitor of GSK-3beta—To directly establish that beta-catenin signaling is associated with an anabolic response in the skeleton, a small molecule inhibitor of GSK-3beta (GSK3betai) was used to stabilize and promote nuclear accumulation of beta-catenin and activation of the Wnt canonical pathway (36). GSK3betai (1 mg/kg/day) was administered to wild type C57BL/6 mice via local subcutaneous injection over the right side of the calvaria. Strong beta-catenin expression was observed in pre-osteoblasts and osteoblastic cells lining the periosteum after 7 days of GSK3betai treatment, indicating that the agent was active in this model (Fig. 3). We also observed significant enhancement of several indices of osteoblast activity including an increase in staining for alkaline phosphatase in MAR and in calvarial thickness (Fig. 4, A and B). These observations suggest that activation of beta-catenin signaling through inhibition of GSK-3beta produces a bone anabolic effect.

Increased Wnt/beta-Catenin Pathway Gene Expression in Response to Mechanical Loading in Wild Type Mice Treated with GSK3betai—Having demonstrated that targets of the Wnt/beta-catenin-signaling pathway are up-regulated by mechanical loading and that an inhibitor of GSK-3beta increased the expression of beta-catenin in osteoblasts and promotes bone formation, we wanted to determine the role of Wnt/beta-catenin pathway activation in the bone formation response to mechanical loading. In vivo loading experiments were performed with mice treated systemically with GSK3betai (50 mg/kg/BID, subcutaneously for 14 days) to activate the canonical Wnt pathway, and gene expression analysis was performed on RNA harvested from the loaded and unloaded tibiae. In these experiments the expression of representative Wnt pathway and Wnt/beta-catenin target genes was measured as well as the expression of the stress responsive genes, COX-2 and eNOS, which served as positive controls to confirm that the tibiae were responding to the loading regimen.

In these in vivo experiments load increased the expression of COX-2 and eNOS 10- and 3-fold, respectively (Fig. 5). Furthermore, load significantly increased the expression of Wnt10B, SFRP1, Cxn43, CCND1, FzD2, and WISP2 ranging from 2- to 4-fold compared with the non-loaded limb. When mice were treated with the GSK3betai for 14 days and then subjected to mechanical loading, there was a further increase (p < 0.01 or p < 0.001) in the expression of stress-responsive genes COX-2 and eNOS as well as Wnt10B, SFRP1, Cxn43, and FzD2 when compared with the loaded tibiae RNA samples from the control mice. The maximal increase in gene expression for COX-2, eNOS, Wnt10B, SFRP1, Cxn43, and FzD2 was 28, 5, 7, 3, 5, and 7-fold respectively (Fig. 5). Changes in CCND1 did not reach statistical significance in response to load in mice treated with GSK3betai. Furthermore, the GSK-3beta inhibitor had no effect alone on the expression of the Wnt/beta-catenin-responsive genes because the transcriptional responses are likely dependent on dose, which may not have been sufficient to elucidate a response alone.


Figure 2
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  		FIGURE 2.
Expression analysis of Wnt pathway and Wnt/beta-catenin target genes in LRP5 G171V transgenic (TG) and non-transgenic mice subjected to mechanical load. Quantitative RT-PCR analysis from both male and female tibia RNA are shown. Panel A demonstrates the responses 4 h post-load. Panel B illustrates the responses 24 h post-load. Transcriptional responses are represented as -fold change (Log2) of loaded responses relative to non-loaded sample responses (*, p < 0.01).

 
Activation of the Wnt/beta-Catenin Pathway Enhances the Sensitivity of Bone Cells to Mechanical Loading—To further investigate these load-induced gene responses in a cellular model, we performed experiments using the Flexercell in vitro loading system (39). MC3T3-E1 cells were subjected to 3400 microstrain for 5 h, resulting in the increased expression of COX-2 (2.5-fold) and eNOS (2.5-fold) as well as c-fos (3.5-fold) and c-Jun (3.5-fold) (p < 0.05), indicating that the cells were responsive to the applied strain (Fig. 6). Consistent with our in vivo loading studies, we also observed increased expression of Wnt10B, CCND1, Cxn43, SFRP1, FzD2 (3-3.5-fold, p < 0.05)), and WISP2 (which was minimal but statistically significant p < 0.05) in strain versus the control cultures.

We next tested whether mechanical strain would synergize with independent activation of the Wnt/beta-catenin pathway by treatment of the cells with either the GSK-3beta inhibitor or the addition of exogenous Wnt3A. When cells were subjected to mechanical strain in the presence of 5 µM GSK3betai, a synergistic transcriptional response of COX-2, eNOS, c-Fos, and c-Jun as well as Wnt10B, CCND1, Cxn43, SFRP1, WISP2, and FzD2 was observed (Fig. 6). The maximum increase in expression for these genes with GSK3betai was 2-3-fold (p < 0.05) above mechanical strain alone. FzD2 and WISP2 demonstrated a 2-fold (p < 0.05) increase in gene expression in the presence of the GSK-3beta inhibitor alone, and although there was little effect on COX-2, Cxn43, Wnt10B, c-Fos, and eNOS expression, it was statistically significant (p < 0.05) (Fig. 6). The GSK-3beta inhibitor alone had no effect on CCND1, SFRP1, or c-Jun expression. We next tested whether a natural canonical Wnt pathway ligand (Wnt3A) that acts through the LRP5 receptor would have similar effects on the strain-induced gene expression as inhibition of GSK-3beta. Treatment of MC3T3-E1 cells with increasing amounts of Wnt3A-conditioned media in the presence of strain resulted in a biphasic, dose-dependent, synergistic increase in COX-2, c-Fos, c-Jun Wnt10B, CCND1, Cxn43, WISP2, and SFRP1 expression compared with mechanical strain (Fig. 7). The magnitude of these responses was 1.8-2.6-fold (p < 0.05) above strain alone. Wnt alone had no effect on the expression of these genes (Fig. 7).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The skeletal phenotype of individuals displaying the LRP5 G171V mutation is quite unique among genetic disorders leading to increased bone density since the bones are predominately of normal shape and size. There have been individuals in one of the kindreds with this mutation, presenting with a mandibular phenotype (2), torus palatinus, or more extreme phenotypes (40), but for most affected individuals the increased density is benign. What is particularly striking about the skeletal phenotype is the fact that the increase in bone density appears to be most evident in load-bearing bones, which has led to the hypothesis that the genetic mutation is acting to augment the skeleton response to mechanical loading (24, 25).


Figure 3
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  		FIGURE 3.
Histological evaluation of calvariae. beta-catenin expression (top panel; magnification 40x), alkaline phosphatase activity (middle panel; magnification 40x), and calcein double labeling for mineral apposition rate assessment (bottom panel; magnification 20x) in vehicle or GSK3betai-treated calvariae of wild type mice.

 


Figure 4
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  		FIGURE 4.
Quantitative analysis of the effects of GSK3betai-treated calvariae in wild type mice represented in Fig. 3. Panel A illustrates the responses on MAR, and panel B demonstrates the responses on calvarial thickness. *, p < 0.05 compared with vehicle-treated samples.

 


Figure 5
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  		FIGURE 5.
Expression of stress-responsive genes, Wnt pathway, and Wnt/ beta-catenin target genes in wild type mice treated with either vehicle or GSK3betai for 14 days and then subjected to mechanical loading. Quantitative RT-PCR was performed on tibia RNA 4-h post-load. Transcriptional responses are represented as -fold change (Log2) of loaded responses relative to non-loaded sample responses. p < 0.01 (*) and p < 0.001 (**) compared with vehicle-treated loaded samples.

 
Once achieved, this high bone density phenotype is maintained as the normal new homeostatic level that is supported by the relatively normal index of mineral metabolism and bone turnover markers (2). The skeletal pattern seen in affected humans is recapitulated in the LRP5 G171V transgenic mice, where bone density levels reach a dramatically elevated plateau that appears to be maintained throughout life. Also, the increased trabecular bone mineral density and trabecular number as well as the slight increase in cortical thickness of LRP5 G171V mice compared with non-transgenic mice is directly associated with enhanced structural and mechanical properties in the femurs and vertebra (5). We have previously shown applying physiological levels of strain (>5000 microstrain is considered pathological (31, 39)) using an established loading paradigm that less mechanical strain is required to stimulate similar bone responses in LRP5 G171V transgenic mice than in non-transgenic littermates (25, 26). Furthermore, we have previously demonstrated that the LRP5 G171V transgenic mice experience significantly less bone loss than non-transgenic mice in a disuse bone un-loading model (41). Collectively, these observations suggest that the LRP5 G171V mutation alters the response of the skeleton to normal mechanical load.

It is interesting to note that we observed no difference in the expression of the osteoblast differentiation transcription factor RUNX2 in the osteoblasts of the LRP5 G171V transgenic compared with non-transgenic mice (data not shown). This suggests that like the low bone mass phenotype in the LRP5 null mice (6), the LRP5 G171V mutation-induced increase in anabolic activities in bone are also RUNX2-independent. In fact our preliminary findings from osteoblasts of LRP5 G171V transgenic animals showing increased expression of Wnt/beta-catenin pathway components and target genes implicates activated canonical Wnt signaling as a key pathway involved in the high bone mass phenotype.

Our data further confirm a growing body of evidence that the up-regulation of the Wnt/beta-catenin-signaling pathway is required for bone formation in response to mechanical loading. We have previously shown that the LRP5 G171V mice are more sensitive to mechanical loading (23). Sawakami et al. (27) demonstrated that LRP5-/- mice fail to form bone in response to in vivo mechanical loading. Clement-LaCroix et al. showed that LiCL, which inhibits GSK-3 is bone anabolic in LRP5-/- mice (29). Hens et al. (28) demonstrated that the Wnt/beta-catenin-signaling pathway is activated by in vitro mechanical loading of primary osteoblasts isolated from the TOPGAL mouse. Also, Lau et al. (42) recently demonstrated that osteoblasts isolated from C57BL/6 mice activate the expression of Wnt pathway genes more robustly in response to mechanical loading compared with C3H/HeJ mice, which have been shown to respond less to loading (43).


Figure 6
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  		FIGURE 6.
MC3T3-E1 cells were treated with vehicle or 5 µM GSK3betai alone or identical treated cultures were subjected to 3400 microstrain. Quantitative RT-PCR was performed on RNA obtained immediately after 5 h of strain. Transcriptional responses of stress-responsive genes, Wnt pathway, and Wnt/beta-catenin target genes are represented as -fold change relative to vehicle samples where no strain was applied. Asterisks indicate genes where expression after strain alone was significantly (p < 0.05) different from no strain in a Tukey multiple comparison, whereas # indicate transcripts for which the effect of strain in the presence of GSK3betai was significantly (p < 0.05) greater than in the absence of GSK3betai in a Tukey multiple comparison.

 


Figure 7
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  		FIGURE 7.
MC3T3-E1 cells were treated with control-conditioned media or various amounts of Wnt3A-conditioned media or identical-treated cultures were subjected to 3400 microstrain. Quantitative RT-PCR was performed on RNA obtained immediately after 5 h of strain. Transcriptional responses of stress-responsive genes, Wnt pathway, and Wnt/beta-catenin target genes are represented as -fold change relative to control-conditioned media samples where no strain was applied. Asterisks indicate doses where the effect of strain (blue) versus the corresponding Wnt3A-matched dose control was significantly larger (p < 0.05) than the effect of strain in the absence of Wnt3A (by Tukey multiple comparison). Data points in red represent non-strained samples.

 
Although our results are similar to those reported by Lau et al. (42) in that we find increased expression of Wnt pathway target genes in response to mechanical loading, they differ in some respects. For instance, we did not observe a significant increase in LRP5 gene expression in our in vivo loading bones, whereas they reported large increases in isolated osteoblasts from the two mouse lines. Several possible explanations may account for this particular difference. First Lau et al. (42) used higher loads in their in vivo loading compared with what we applied (9N versus 6N). Also, they used younger mice (8 weeks versus 17 weeks of age), and hence, the skeletons of the mice in their study were still growing. Finally, it is possible that this difference reflects differences in the C57BL/6 mice from different vendors. Regardless, our results are in general agreement with those of Lau et al. (42) in that mechanical loading does result in the up-regulation of the Wnt/beta-catenin-signaling pathway. Thus, it appears that the regulation of individual components of the Wnt/beta-catenin-signaling pathway may vary depending upon the model, and as such, it will be important to sort out both similarities and differences in terms of understanding the mechanism leading to this response. This is particularly true when attempting to interpret data from cell culture studies using cell lines in comparison with in vivo results where the interactions of multiple cell types are involved in the response to mechanical loading.

There is accumulating evidence from the cancer literature that cross-talk between various signaling pathways such as the prostaglandin (44), bone morphogenetic protein (45), and the Pten tumor suppressor (46) may regulate beta-catenin through inhibition of GSK-3beta. If similar mechanisms function in bone then interpreting the cellular response to mechanical loading requires an integration of multiple signals and understanding the temporal sequence of events that result.

In light of our studies and those of other investigators, how does Wnt/beta-catenin signaling fit into the mechanism by which bone responds to mechanical loading? We have shown that known stress response genes such as COX-2, eNOS, c-Fos, and c-Jun are up-regulated by mechanical loading consistent with the work of others. PGE2 and NO release are some of the earliest changes measured in response to mechanical loading of bone. We believe that these events are upstream of the activation of the Wnt/beta-catenin-signaling pathway. Recent work suggested that PGE2 can lead to a Wnt-independent activation of the canonical beta-catenin-signaling pathway by inhibition of GSK-3beta and binding of G{alpha}s to axin (44). Furthermore, Sawakami et al. (27) showed that PGE2 release from LRP5-/- osteoblasts by fluid sheer stress was not affected by loss of LRP5 expression. This may suggest that cross-talk between PGE2 signaling and Wnt/beta-catenin signaling could occur in response to mechanical loading independent of LRP5. So we directly tested the effects of activating Wnt/beta-catenin signaling on in vivo bone formation using GSK3betai and found a bone anabolic response that was equivalent to that generated by exogenous administration of PTH (data not shown).

Having shown a bone anabolic effect with GSK3betai, studies were designed to determine whether the effects of mechanical loading on gene expression in wild type mice treated with GSK3betai would mimic that in LRP5 G171V transgenic mice where the mutation is a canonical Wnt pathway activating mutation. Interestingly, we observed similar increases in the expression of the Wnt/beta-catenin target genes in the GSK3betai-treated non-transgenic mice subjected to load compared with the LRP5 G171V transgenic mice. These data strongly suggest that activation of beta-catenin signaling rather than other non-canonical signaling pathways enhance the effects of mechanical loading on bone. These observations are supported by the mechanical loading experiments where MC3T3-E1 cells treated with increasing amounts of either GSK3betai (data not shown)- or Wnt3A (a canonical Wnt pathway ligand)-conditioned media caused a dose-dependent synergistic up-regulation of Wnt/beta-catenin gene expression. Interestingly, high concentrations of exogenous Wnt3A resulted in a down-regulation of these genes. This biphasic transcriptional response observed with Wnt3A (that was not seen with GSK3betai) may have been due to negative feedback regulation at the level of the LRP5/FzD co-receptor that may be different with GSK3betai, which acts downstream of this co-receptor complex. Implicit in our model is a feedback regulation of the Wnt/beta-catenin-signaling pathway. If initial activation of beta-catenin results from cross-talk with other pathways at the level of GSK-3beta, then sustained activation requires the expression of genes whose protein products (such as Wnts, Frizzles, etc.) will amplify the initial load activation and commit the cells to new bone formation. This could explain why LRP5-/- mice fail to form bone in response to loading because the feedback amplification cannot occur when LRP5 is absent.

Collectively, the in vivo and in vitro mechanical loading results suggest that Wnt/beta-catenin signaling is a normal physiological response to load and that activation of the Wnt/beta-catenin pathway enhances the sensitivity of bone cells to mechanical loading. The HBM phenotype shows an enhanced anabolic response to loading on bone that may be due to an increase in osteoblast differentiation and function and/or reduced inhibition of LRP5 (2-4), enabling a longer sustained activation of the pathway in response to load. In fact we find genes important for such functions including c-Fos, c-Jun (AP-1), eNOS, COX2 (involved in PGE2 production), and Cxn43 (47-52) all up-regulated to a greater extent with mechanical loading when the canonical Wnt pathway was activated. Furthermore, it is also possible that the cooperative actions of mechanical loading and activation of the canonical Wnt pathway promote osteoblastogenesis because an induction in expression of Wnt10B an inhibitor of adipogenesis (53, 54) was also observed.

The fact that a combination of pharmacologic treatment with a GSK-3beta inhibitor and mechanical loading produce a synergistic effect has important clinical implications. Concerns over the safety of therapies designed to focus on regulating the Wnt signaling pathway has been debated in the literature (11, 55, 56). If a combination of exercise (loading) and a otherwise sub-efficacious dose of pharmaceutical could be used to generate a bone anabolic response, this novel approach might offer great hope for individuals afflicted with diseases of low bone mass such as osteoporosis. Much more work in this regard is needed, but our data suggest this as a real possibility.

In summary, we have used both genetic and pharmacological models to show the involvement of the canonical Wnt pathway on mechanical loading responses in the skeleton. The in vitro and in vivo data corroborate the conclusion that mechanical loading regulates Wnt pathway and Wnt/beta-catenin target gene expression and that Wnt signaling enhances the sensitivity of bone cells to mechanical loading. These findings demonstrate a novel role for canonical Wnt signaling in adult bone mass regulation. It is conceivable that mimicking the positive effects of the HBM LRP5 G171V mutation on the skeleton pharmacologically may provide novel therapies for the treatment of osteoporosis.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

2 Both authors contributed equally to this manuscript. Back

3 Present address: Aventis, 1041 Rt. 202/206, N303A, Bridgewater, NJ 08807. Back

4 Present address: Dept. of Microbiology, School of Medicine, University of Washington, Seattle, WA 98195. Back

5 Present address: Amgen, Inc, 1 Amgen Center Drive, Thousand Oaks, CA 91320. Back

6 Present address: Dept. of Oral Biology, Kansas City Dental School, Kansas City, MO 64108. Back

1 To whom correspondence should be addressed: Dept. Women's Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Rd., RN3247, Collegeville, PA 19426. Tel.: 484-865-2850; Fax: 484-865-9395; E-mail: robinsj{at}wyeth.com.

7 The abbreviations used are: GSK-3beta, glycogen synthase kinase-3beta; HBM, high bone mass; PG, prostaglandin; MAR, mineral apposition rate; SFRP1, secreted frizzled-related protein 1; WISP2, Wnt1-inducible signaling pathway protein 2; eNOS, endothelial nitric-oxide synthase; Cxn43, connexin 43; FzD2, frizzled 2; CCND1, cyclin D1. Back

8 Microstrain is defined as a unit of strain that is the percentage of change in length or relative deformation (10,000 microstrain = 0.01 strain = 1% deformation). Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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S. Kang, C. N. Bennett, I. Gerin, L. A. Rapp, K. D. Hankenson, and O. A. MacDougald
Wnt Signaling Stimulates Osteoblastogenesis of Mesenchymal Precursors by Suppressing CCAAT/Enhancer-binding Protein {alpha} and Peroxisome Proliferator-activated Receptor {gamma}
J. Biol. Chem., May 11, 2007; 282(19): 14515 - 14524.
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P. V. Bodine
Wnt Signaling in Bone
IBMS BoneKEy, March 1, 2007; 4(3): 108 - 123.
[Abstract] [Full Text] [PDF]

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